Institute of Animal Husbandry and Animal Breeding University of

Transcrição

Institute of Animal Husbandry and Animal Breeding
University of Hohenheim
Prof. em. Dr. Dr. habil. R. Claus
Effects of immunological castration on the regulation of metabolism in boars
DISSERTATION
submitted in fulfilment of the requirements for the degree “Doktor der Agrarwissenschaften“
(Dr.sc.agr./ Ph.D. in Agricultural Sciences)
to the
Faculty of Agricultural Sciences
presented by
Aneka Bauer
Heilbronn
Hohenheim 2010
This thesis was accepted as a doctoral dissertation in fulfilment of the requirements for the
degree “Doktor der Agrarwissenschaften” by the Faculty of Agricultural Sciences of the
University of Hohenheim on 29. September 2010.
Date of oral examination: 11. October 2010
Examination committee
Supervisor and Reviewer
Prof. Dr. Dr. R. Claus
Co-Reviewer
Prof. Dr. Dr. H. Sauerwein
Additional examiner
Prof. Dr. R. Böhm
Vice-Dean and Head of the Committee
Prof. Dr. A. Fangmeier
Meinen Eltern
Table of Contents
Table of Contents
1
Introduction
7
1.1
Tradition of castration in pig production
7
1.2
Advantages and disadvantages of fattening entire male pigs
7
1.3
Chemical aspects of boar taint
10
1.3.1
Androstenone
11
1.3.2
Skatole
15
1.4
Anabolic effects of testicular hormones
16
1.4.1
Physiological principles
16
1.4.2
Attempts to abolish androstenone but to maintain the anabolic
17
potential
1.5
Animal welfare and castration
19
1.6
Principles of immunization and its effects on metabolism
21
1.7
Theories underlying the own experimental studies
24
1.7.1
Interaction between the releasing hormones GnRH and GH-RH
24
in the hypothalamus or the pituitary
1.7.2
Influence of different feed intake on GH and IGF-I levels
25
1.7.3
Imprinting of a male specific GH secretion pattern
26
2
Own Experimental Studies
28
2.1
Short-term endocrine and metabolic reactions before and after
28
second immunization against GnRH in boars
(R Claus, M Lacorn, K Danowski, MC Pearce, A Bauer,
Vaccine 2007, 25: 4689-4696)
2.2
Effects of immunization against GnRH on gonadotropins, the
38
GH-IGF-I-axis and metabolic parameters in barrows
(A Bauer, M Lacorn, K Danowski, R Claus,
Animal 2008, 2: 1215-1222)
2
Table of Contens
2.3
Effects of two levels of feed allocation on IGF-I concentrations
48
and metabolic parameters in GnRH-immunized boars
(A Bauer, M Lacorn, R Claus,
Journal of Animal Physiology and Animal Nutrition 2009, 93:
744-753)
2.4
Is the early postnatal rise of testosterone responsible for a later
60
male pattern of growth hormone secretion in pigs?
(M Lacorn, A Bauer, R Claus,
Theriogenology 2009, 72: 636-642 )
3
General Discussion
68
4
Summary
72
5
Zusammenfassung
75
6
References
78
7
Curriculum Vitae
94
3
Table of Contents
Tables and Figures
Table 1a:
Differences between the sexes during fattening from 40-110 kg
8
body weight (Witt and Schröder, 1969)
Table 1b:
9
body weight (Campbell et al., 1989)
Table 1c:
9
body weight (Spring et al., 2009)
Table 2:
Percentage of animals in androstenone classes influences by
10
sex (Stamer et al., 1993)
Table 3:
Ability to smell androstenone
15
Table 4:
GH concentrations (ng/mL) of entire boars, immunized boars,
24
and barrows (Metz, 2003)
Figure 1:
Compounds contributing to boar taint (Claus, 2005)
11
Figure 2:
Biosynthesis of gonadal steroids (Bonneau, 1982)
12
Figure 3:
Synthesis of androstenone in the testes and its release via the
13
salivary gland (Claus, 1991)
Figure 4:
Time course of pubertal development of boars (Claus et al.,
14
1994)
Figure 5:
Schematic view of skatole formation in the colon (Weiler et al.,
15
1992)
Figure 6:
Principles of endocrine regulation of protein deposition (Claus
17
et al., 1994)
Figure 7:
Neuro-endocrine structures relevant for “immunological
22
castration” (Rosenzweig et al., 1996)
Figure 8:
Hypothetical interaction between GnRH and GH-RH in barrows
25
and immunized barrows
Figure 9:
GH-IGF-I system and regulative parameters (Claus, 2005)
25
4
Tables and Figures
Figure 10:
Early and late castration in the time course of pubertal
26
development of boar testes function
Figure 11:
Weight development of immunized boars between 18 and 28
weeks of age (dashed lines mark possible slaughter weights
and the corresponding age; arrows mark second vaccination
relative to slaughter, 2. IM)
5
69
Table of Contents
Abbreviations
DHT
5α-dihydrotestosterone
FSH
Follicle stimulating hormone
GCR
Glucocorticoid receptor
GH
Growth hormone
GH-RH
Growth hormone releasing hormone
GnRH
Gonadotropin releasing hormone
HSD
3β-hydroxysteroid dehydrogenase
IGF-I
Insulin-like growth factor I
LH
Luteinizing hormone
6
Introduction
1
Introduction
1.1
Tradition of castration in pig production
Surgical castration of male animals is one of the oldest biotechnical measures applied in
animal production and has been known to almost all nations and cultures. The primary
reason was the abolishment of testicular hormones and thus their stimulating effect on
aggressive behaviour. In consequence, the castrated males were easier to handle.
The tradition to castrate male pigs goes back to medieval times and was additionally
performed because the castrated male pigs (barrows) deposit much more fat instead of
protein than entire male pigs (boars). Fat accumulation was preferred by the market until a
couple of decades ago because its high energy content was required by a hard working
human population (EFSA, 2004). When muscle work was substituted by machines and
automation in industry started about 60 years ago, this process was paralleled by a rapid
change of market requirements toward carcasses with less fat and more muscle protein.
Today, carcasses which contain maximal amounts of lean meat are definitely preferred. In
consequence, discussions arose to leave boars uncastrated because their carcasses are
characterized by high deposition of protein and accordingly decreased accumulation of fat.
But the main cause of still castrating male pigs is the occurrence of the so-called boar taint in
the carcass of entire male pigs. This undesirable urine-like odour is not acceptable to many
consumers (Matthews et al., 2000). Nevertheless, fattening of entire male pigs is still under
discussion, hoping that the formation of anabolic hormones and boar taint, which are both
synthesized in the testes, may be separated some day.
1.2
Advantages and disadvantages of fattening entire male pigs
Many studies confirmed that differences in fattening performance exist between castrates
(barrows), intact males (boars) and females (gilts) (Witt and Schröder, 1969; Walstra, 1974;
Campbell et al., 1989; Neupert et al., 1995) which are explained by the availability of the
anabolic gonadal hormones. However, intensive breeding for protein deposition led to a
remarkable shift in the meat-fat-ratio so that barrows today have the same fattening
performance as reported for boars 25 years ago (Claus, 1993). Nevertheless, the influence
of gonadal steroids still exists and thus the differences between the sexes.
Economic advantages may result when leaving boars uncastrated and useing them routinely
for meat production. These advantages include lower production costs, e.g. no labour costs
resulting from castration, the absence of animal losses, and a decrease in growth
7
Introduction
performance following castration. In addition, entire boars grow more rapidly and exhibit an
improved feed conversion rate (Table 1a-c). In a study by Spring et al. (2009; Table 1c),
barrows had a higher daily weight gain and a higher daily feed consumption. But this
supposed better performance resulted in fat deposition instead of lean meat, which is
underlined by the thickness of back fat and the less lean meat content. Additionally, barrows
in this study had a worse feed conversion rate compared with boars. Thus, boars produce
carcasses with higher retail yields due to the fact that they synthesize more lean meat and
less fat than their castrated counterparts (Martin, 1969; Ellis et al., 1983; Dobrowolski et al.,
1993). Gilts usually are intermediate in these criteria while barrows are generally inferior in all
these traits (Walstra and Kroeske, 1968; Kay and Houseman, 1975).
Further boars have the ability of a higher protein retention which is caused by the
anticatabolic effect of gonadal steroids. Therefore, the nitrogen excretion is reduced. In areas
with a high production density, the fattening of boars was considered as a possible
alternative to avoid higher pollution of the soil and drinking water with nitrogen, but to
maintain or even increase the number of animals per production unit (Claus, 1993). In
studies where barrows were treated with a combination of androgens and oestrogens, the
nitrogen retention was increased by 25%. (Van der Wal et al., 1975; Van Weerden and
Grandadam, 1976; Berende et al., 1980). Furthermore, the treatment with androgens and
oestrogens resulted in an average increase in protein deposition of 40%, and fat deposition
was decreased by 15-20%. These results confirm that gonadal steroids affect the balance
between protein synthesis and breakdown in favour of a higher protein deposition (Van
Weerden and Grandadam, 1976). The anticatabolic effect of androgens, and thus the
improved N-retention again, is explained at least partly by the fact that androgens antagonize
glucocorticoids and their catabolic functions (Mayer and Rosen, 1977; Snochowski et al.,
1981).
Table 1a:
Differences between the sexes during fattening from 40-110 kg body weight
(Witt and Schröder, 1969)
boars
barrows
gilts
feed consumption (kg/d)
2.45
2.54
2.48
weight gain (g/d)
808
727
724
feed conversion rate
3.05
3.51
3.45
back fat thickness (cm)
3.12
3.53
3.32
8
Introduction
Table1b:
(Campbell et al., 1989)
boars
barrows
gilts
3.21
3.67
3.38
weight gain (g/d)
1186
1057
1011
2.72
3.46
3.34
1.48
2.50
2.12
Table 1c:
(Spring et al., 2009)
boars
barrows
2.06
2.35
weight gain (g/d)
885
927
2.33
2.54
1.29
1.95
lean meat content (%)
56.6
53.2
An argument against routine fattening of boars is an inferior meat quality. Meat of boars can
be too lean and thus is often found to be less tender, and meat with a deficit in intramuscular
fat is inferior in flavour and juiciness (Branscheid, 1993). Similarly, the processing industry
needs a minimum of fat content, but the fat of entire males is softer and less resistant to
oxidation due to a higher quantity of unsaturated fatty acids (Malmfors and Nilsson, 1978;
Barton-Gade, 1987). In addition to these criteria, the most eminent limitation to use entire
male pigs is the occurrence of boar taint.
9
Introduction
1.3
Chemical aspects of boar taint
Consumer acceptance of pork from boars is hindered by a strong, objectionable odour of the
heated fat and meat of many boars (Malmfors and Lundström, 1983; Matthews et al., 2000).
Table 2 shows the percentage of pigs separated in androstenone classes. 87% of the boars
had androstenone concentrations higher than 0.3 µg/g fat, whereas all barrows were below
this value. This “perspiration-like” or “urine-like” odour has become known as “boar odour”,
“boar taint” or “male sex odour” in pork and is associated with the ∆16-steroid 5α-androst-16en-3-one (androstenone; Patterson, 1968). More recently, 3-methylindole (skatole, Void
1970) with a faecal odour was suspected as another reason for “boar taint”, which stimulated
discussions on the relative contribution to the unpleasant odour (Bonneau, 1982).
Table 2:
Percentage of animals in androstenone classes influenced by sex (Stamer et
al., 1993)
androstenone level
barrows
boars
(µg/g fat)
(%)
(%)
< 0.04
86.5
10.3
0.04-0.3
13.5
2.6
0.3-1
0
59.0
>1
0
28.1
Figure 1 compares typical characteristics of the two compounds. Androstenone is a steroid
which has a chemical structure similar to the testicular sex hormones, but it has no hormonal
activity (Claus, 1979).The characteristic double bond for the ∆16-steroids at the C-16 results
in smell properties of androstenone instead of hormonal activity. In contrast, skatole is
derived from the amino acid trypthophan and is not directly linked to testicular function.
10
Introduction
androstenone
skatole
chemistry
steroid
indole
site of synthesis
testes
colon
urine-like odour
faecal-like odour
not selectively
influencable
influencable by
nutrition
type of smell
possibilities to inhibit
the formation
Figure 1:
1.3.1
Compounds contributing to boar taint (Claus, 2005)
Androstenone
Androstenone is synthesized in the Leydig cells of the testes in parallel to the synthesis of
anabolic sex hormones (Gower and Ahmad, 1967) which in the porcine species comprise
androgens and oestrogens (Claus and Hoffmann, 1980). Synthesis of androstenone and of
testicular hormones is stimulated by the luteinizing hormone (LH), which in turn is stimulated
by gonadotropin releasing hormone (GnRH) (Andresen, 1975; Carlström et al., 1975). Thus,
the degree of boar taint is strictly coupled to the degree of formation of anabolic hormones.
The occurrence of a group of steroids with smell properties (“16-unsaturated steroids” or
∆16-steroids) in testicular tissue from boars was first described by Prelog and Ruzicka
(1944) who also reported on the urine-like taint of androstenone due to the presence of a
ketogroup at the C-3 position. This substance was later found to be present in high
concentrations in adipose tissue and to be the reason for the characteristic urine-like boar
taint (Patterson, 1968; Claus et al., 1970; Claus et al., 1971; Beery and Sink, 1971; Fuchs,
1971; Thompson et al., 1972).
11
Figure 2:
progesterone
androstadienone
5α-androst-16-en-3-one
pregnenolone
androstadienol
cholesterol
17α-hydroxyprogesterone
17α-hydroxypregnenolone
estrone
androstenedione
dehydroepiandrosterone
17β-estradiol
testosterone
androstenediol
Introduction
Biosynthesis of gonadal steroids (Bonneau, 1982)
12
Introduction
The principle of biosynthesis of androstenone together with the other testicular steroids is
summarized in Figure 2. LH stimulates the synthesis of the precursor steroid pregnenolone
which then is delivered by the enzyme 3β-hydroxysteroiddehydrogenase (HSD) both for
further synthesis of the gonadal hormones (androgens and oestrogens) and the ∆16-steroids
(androstenone). Thus a close relationship exists between the secretion of androstenone
(boar taint) and the sexual status of the male pig. Many attempts to suppress androstenone
selectively but to maintain the anabolic potential of boars have been unsuccessful due to the
common biosynthesis (see 1.4.2).
Figure 3:
Synthesis of androstenone in the testes and its release via the salivary gland
(Claus, 1991)
Androstenone is transferred from the testes into the blood circulation via the vena spermatica
(concentrations of 20 ng/mL plasma; Andresen, 1974; Claus and Hoffmann, 1980). Due to its
lipophilic character, androstenone is accumulated in adipose tissue. The adipose tissue acts
like a reservoir and androstenone can be released from fat when de novo synthesis is
decreased in the testes (Figure 3). Claus (1979) found androstenone concentrations of 0.2-4
µg/g fat in carcasses with 80 kg of weight. Neupert et al. (1995) analysed carcasses with 85
kg of weight and determined mean androstenone values of 1.21 µg/g fat. The high variation
of androstenone concentrations is due to differences in the stage of pubertal development
which is reached during the final period of fattening and depends e.g. on breed. The final
pubertal spurt is expected at an age of 15-17 weeks (Figure 4) (Booth, 1975; Allrich et al.,
1982; Bonneau, 1987; Schwarzenberger et al., 1993). In addition, the variability of
androstenone levels is influenced by housing conditions (e.g. groups of boars and thus social
interactions) (Giersing et al., 1997, 2000).
Physiological androstenone acts as a pheromone during mating behaviour and induces the
standing reflex of the sow before copulation (Signoret et al., 1960; Melrose et al., 1971;
13
Introduction
Willems, 1972; Reed et al., 1974). Additionally, it may lead to the release of oxytocin as a
stimulant of sperm transport during mating (Claus and Schams, 1990).
testes function
(testosterone, androstenone)
2-5
Figure 4:
15-17
age (weeks)
Time course of pubertal development of boars
(Claus et al., 1994)
The sensibility of consumers for androstenone differs individually. Some persons have a
specific anosmia for androstenone, i.e. they are not able to smell androstenone although
these persons have a normal sense of smell for other substances (Table 3). Among the
persons who are able to smell androstenone, some are highly sensitive even to
concentrations below the threshold of 0.5 µg/g fat. Another limitation for the organoleptic
evaluation of androstenone is that even persons with the ability to smell this substance can
judge negative samples as positive ones because after smelling androstenone, the receptors
are occupied for a certain time (“reverberation effect”).
14
Introduction
Table 3:
Ability to smell androstenone
persons
none
less
medium
strong
(n)
(%)
(%)
(%)
(%)
286
25.0
23.5
33.5
18.0
Elsley, 1968
301
27.5
-
-
17.5
Griffith and Patterson, 1970
520
28.5
22.0
29.0
20.5
Claus unpublished
1.3.2 Skatole
Skatole is produced by microbial degradation of the amino acid tryptophan in the colon
(Figure 5). A part of skatole is excreted with faeces but another part is resorbed from the gut
and then accumulated in fat tissue. Neupert et al. (1995) found mean skatole concentrations
of 131 ng/g fat but also a considerable variation between individuals. The faecal odour of
skatole, which is smelled by almost all persons, can be found in barrows as well as in gilts,
but the frequency of high concentrations above a cut-off value of 200 ng/g fat is clearly
higher in entire boars. The presence of high levels in pig meat certainly is an olfactoric
problem, but this off odour can be controlled with feeding strategies. Feeding e.g. potatoe
starch during the last week before slaughter decreases skatole concentrations far below the
cut-off value (Claus et al. 1994, 2003; Lösel and Claus, 2005; Lösel et al., 2006; Spring et al.,
2009).
carcass fat
trypthophan
skatole
diet
faeces
micro organisms
Figure 5:
15
Schematic view of skatole formation in the colon (Weiler et al., 1992)
Introduction
1.4
Anabolic effects of testicular hormones
1.4.1
Physiological principles
In boars, testosterone and 5α-dihydrotestosterone (DHT) are the most important androgens
produced in testes. DHT was measured with maximum concentrations of 2.75 ng/mL in blood
plasma (average concentrations of 0.75 ng/mL). It stimulates specifically reproductive
phenomena such as the development and function of accessory glands (Parrot and Booth,
1984). Testosterone occurs in average plasma concentrations of 2.15 ng/mL (maximum of
11.50 ng/mL; Claus et al., 1983). The variation in androgen concentration depends on the
time of day (Claus and Gimenez, 1977; Claus and Hoffmann, 1980), on age (Lapwood and
Florcruz, 1978; Allrich et al., 1982; Schwarzenberger et al., 1993) and on the seasonal effect
of the photoperiod (Hoagland and Diekmann,1982; Claus et al., 1983; Andersson et al.,
1997).
In addition to its role for reproduction, testosterone is involved in the stimulation of male
behaviour including aggression, and it exerts various effects on metabolism. The latter
effects are mediated by androgen receptors in muscle tissue (Snochowski et al., 1981) and
include both a stimulation of protein synthesis and deposition (“anabolic effect”; Claus and
Weiler, 1994) and an inhibition of catabolic effects provided by glucocorticoids (“anticatabolic
effect”; Mayer and Rosen, 1977; Sharpe et al., 1986; Chen et al., 1997). This anticatabolic
effect is mainly responsible for the improved protein deposition found in entire boars
compared to barrows and, less pronounced, compared to gilts (Metz, 2003).
Boars additionally synthesize high amounts of oestrogens such as 17β-oestradiol. Its
concentrations in boar blood plasma may even exceed levels found in oestrus sows (Claus
and Hoffmann 1980; Claus et al., 1983, 1987). Oestrogens have synergistic effects to
androgens, e.g. for male behaviour and libido (Parrot and Booth, 1984), and also for
spermatogenesis (Wagner et al., 2006). Oestrogens also have an effect on growth because
they stimulate protein deposition indirectly by stimulating the GH-IGF-I system (GH growth
hormone, IGF-I insulin-like growth factor I) (Claus and Weiler, 1994). For this role,
oestrogens lead to a rise of IGF-I by increasing the expression of GH receptors in the liver
(Gabrielsson et al., 1995). In consequence, normalization of a boar-specific growth potential
by steroid application in barrows requires a combination of an androgen and an oestrogen
(Van Weerden and Grandadam, 1976; De Wild and Lauwers, 1984) as outlined in Figure 6.
16
Introduction
GCR
inhibit
androgens
GH
IGF-I
anabolic status
oestrogens
Figure 6:
Principles of endocrine regulation of protein deposition (Claus et al., 1994)
(GCR glucocorticoid receptor)
Producers should take advantage of the anabolic hormones produced by the testes. But the
close relationship between synthesis of anabolic steroids and of androstenone makes the
success of pork production with intact boars doubtful. Attempts to separate the secretory
capabilities of the testes into anabolic hormones and pheromones failed so far.
1.4.2 Attempts to abolish androstenone but to maintain the anabolic potential
Many attempts were made to use the metabolic advantages of boars but to abolish boar taint
selectively. They were all directed towards an elimination of androstenone from the carcass
because it is the main substance responsible for the urine taint in almost every boar
slaughtered beyond puberty.
One possibility was the “hormonal castration” and thus the control of androstenone synthesis
by exogenous application of hormones (Echternkamp et al., 1969; Ertl et al., 1972). This
treatment influenced the secretion of LH causing an atrophy of the Leydig cells and
decreased testicular activity (Sheridan et al., 1990). In consequence, androstenone as well
as testosterone were reduced, i.e. the anabolic potential was also lost in treated boars
(Daxenberger et al., 2001). The implantation of anabolic preparations into barrows increased
nitrogen retention and decreased fat deposition (Van Weerden and Grandadam, 1976; De
Wild and Lauwers, 1984). Therefore, surgical castration together with application of anabolic
hormones can improve the fattening performance of barrows. But these attempts are
theoretical possibilities because they have no practical relevance due to residue
considerations and consumer protection.
Genetic selection for pigs with low levels of tissue androstenone concentrations at the time of
slaughter is possible (Willeke et al., 1980; 1987) and a high heritability was confirmed. But
17
Introduction
these attempts also resulted in a decreased performance due to joint testicular biosynthesis
of anabolic steroids and androstenone which could not be decoupled. The genetic selection
also led to a delay of puberty in boars as well as in gilts. Therefore, genetic selection for low
androstenone concentrations has a negative influence on reproductive traits which is not
desirable for breeding.
Another approach was the active immunization against androstenone which was basically
successful (Claus, 1975; Williamson et al., 1985; Brooks et al., 1986). However, this
possibility was not investigated further because titre development differed considerably
between individuals and repeated immunization injections were required.
An alternative could be to sort X from Y carrying sperm so that only X-sperm are used for
artificial insemination to produce only gilts for fattening. But this method is currently only in a
state of research, i.e. this strategy is far from practical application and is not paralleled with
the higher anabolic potential of boars. One problem is the huge number of sperm needed for
a satisfactory insemination with the ordinary insemination technique (Johnson et al., 2005;
Martinez et al., 2005). In addition, sorting of sperm would considerably rise the costs of pig
production.
Slaughter at a young age before the final pubertal spurt and thus less probability of
occurrence of androstenone is an alternative which has traditionally been performed in
England and explains the acceptance of boar meat by part of the consumers. Today,
economic considerations require slaughter of pigs with much higher and still increasing final
weights. Therefore, the number of tainted carcasses from entire boars will inevitably rise.
Processing of meat by procedures which remove androstenone from the product or destroy
the chemical structure of the substance could be a possible solution to use pork from entire
males. Claus et al. (1985) found that almost all of the traditional products still contain the
levels of androstenone as measured in raw material before processing. Only products which
were subject to prolonged heating may have partially reduced androstenone concentrations
due to evaporation. Another study investigated the possibility to overlay boar taint with
flavours from fermentation or smoking (Stolzenbach et al., 2009). Mixing of processed meat
with spices revealed no satisfactory results (Lunde et al., 2008).
As there are no alternatives to inhibit boar taint, surgical castration is performed in most
countries up to now. It is performed usually without anaesthesia early after birth. Since a
couple of years, however, surgical castration without anaesthesia became an animal welfare
issue so that alternatives are urgently required.
18
Introduction
1.5
Animal welfare and castration
Every year approximately 100 million male piglets are castrated without anaesthesia in the
European Union (Thun et al., 2006; Heinritzi et al., 2008). Due to economical priorities and
lack of suitable alternatives, the animal welfare legislation in most European countries allows
castration up to seven days without anaesthesia. If castration is practiced after seven days of
age, it shall only be performed under anaesthesia and additional prolonged analgesia by a
veterinarian. This practice was also confirmed by the EU Commission Directive
(2001/93/EC).
Castration without anaesthesia is meanwhile regarded to induce acute pain and stress and
will therefore not be tolerated any longer by animal welfare organizations. Piglets that were
not anaesthetized during castration produced a higher proportion of screaming sounds,
which probably indicates acute pain provoked by castration (Puppe et al., 2005). Llamas
Moya et al. (2008) confirmed that the behaviour of piglets is modified after castration. During
the initial hours following castration, pigs showed less locomotion, more trembling and
spasms, they huddled-up more and scratched their rump. Three days later, castrated piglets
were more isolated, showed less social interactions and increased dog sitting behaviour.
Thus, welfare organizations demand a method that is more in accordance with the animal
welfare issue. In Norway, surgical castration without anaesthesia has not been allowed since
2003. Until another solution is found, only veterinarians may perform castration under
anaesthesia. In Switzerland, since 2010 castration has been permitted under anaesthesia
only. The Netherlands are considering to stop surgical castration at the latest by 2015, on the
basis of increasing reactions from consumers in relation to the daily castration routine.
Sweden will prohibit castration in 2012.
Apart from the boar taint problem, raising uncastrated boars is not an optimal alternative
because of fighting between boars and mounting behaviour resulting in leg injuries,
lameness and skin lesions especially towards the end of the fattening period as the boars
become sexually mature (EFSA, 2004). Some studies indicate that aggression in entire
males may be a welfare problem as well (Rydhmer et al., 2006; Salmon and Edwards, 2006;
Boyle and Björklund, 2007; Fredriksen and Hexeberg, 2008; Tuyttens et al., 2008). This
problem will be intensified by split marketing. Fredriksen et al. (2008) found reduced levels of
aggression lesions shortly before slaughter if groups stayed together from birth to slaughter
without any mixing. Some countries as the United Kingdom, Ireland and Spain, which raise
entire males but slaughter at a lower age, gradually increase the age for slaughter on the
grounds that producers earn more when slaughtering heavier pigs. Therefore, the problem
with fighting and boar taint will continuously become more evident in these countries.
19
Introduction
One possible alternative is the castration under general or local anaesthesia followed by
analgesia. The narcotic treatment permits to perform the castration of the piglets without
feeling pain and therefore the welfare aspect is guaranteed. Problems with anaesthesia are
that at the moment only veterinarians are allowed to administer these narcotics, introducing
unacceptable costs for the farmers. The anaesthetic gas isoflurane is currently under
discussion but it additionally requires an analgesic drug to suppress pain the days following
castration (Schulz, 2007; Schulz et al., 2007a, 2007b). In Switzerland, routine castration is
practised under isoflurane inhalation anaesthesia with a specially designed application
system, combined with additional pain treatment before castration. Kupper et al. (2008)
found that 92% of the castrated piglets showed no defence movement.
Another anaesthetic gas used for general anaesthesia is CO2. A mixture of 30% oxygen and
70% CO2 has been found to be the most effective combination (Kluivers-Poodt et al., 2007;
Gerritzen et al., 2008). This anaesthesia is applied in the Netherlands. But CO2 exposure has
been reported to cause distress until loss of consciousness (EFSA, 2004; Rodrigez at al.,
2008), and the anaesthetic and the lethal dose are close together.
Lahrmann et al. (2006) investigated general anaesthesia by an injection method. They found
that anaesthetics are easily given with an injection pistol and that analgesia is satisfactory.
But they reported losses of 3-5%. Other studies showed that the same anaesthetic was
insufficient to induce adequate depth of anaesthesia in a significant number of piglets (Leeb
et al., 2008; Schmidt and von Borell, 2008). It was demonstrated that pigs showed pedal
reflex, vocalisation and withdrawal movements.
In Germany, it was recommended to perform castration with an analgesic agent
(“Düsseldorfer Erklärung”, 2008). An injection of lidocaine into the testis or the spermatic
cord was considered effective in reducing pain during and after the castration (Heinritzi et al.,
2006; Zöls et al., 2006a, 2006b). In a study of Fredriksen and Nafsted (2006), 54% of the
veterinarians and 19% of the producers evaluated the effect of predominantly subcutaneous
and then intratesticular injection of lidocaine with adrenaline to be satisfying. The most
common complications with local anaesthesia reported in this study were abscesses. A
general problem of castration with anaesthesia is its need for a waiting time between
application and the onset of its pain-suppressing effect, as well as the problem that the use
of an anaesthetic can hardly be controlled under routine practice by the farmers. Therefore,
the question arises if surgical castration under anaesthesia is a practical method for the daily
routine in pork production.
Another approach which will not compromise animal welfare is the active immunization
against GnRH. Immunization is a well established technique in animal production, and a
promising and realistic alternative to surgical castration.
20
Introduction
1.6
Principles of immunization and its effects on metabolism
Active immunization against substances produced naturally in the body has been known
since the 1930s (Scaramuzzi et al., 1993). It is an effective method which can abolish
individual endocrine signals so that their role for regulation of physiological phenomena can
be studied in detail. Specifically abolishment of GnRH by active immunization was an early
and promising approach because GnRH is on top of a complex system which influences the
complete reproductive system, i.e. the gonadotropins and the gonadal steroids (Hage-van
Noort et al., 1992; D’Occhio, 1993). Moreover, GnRH is synthesized only in tiny amounts in
the hypothalamus, so that these low amounts can easily be inactivated by antibodies. In
consequence, immunization against GnRH has proven to be a powerful tool for practical, onfarm applications as well as for studying basic aspects of reproduction in the field of
reproduction research (Thompson, 2000). As GnRH is a hapten, which for its own is too
small to be immunogenic, it is chemically linked to a carrier protein. This complex is mixed
with an adjuvant to form a dose for vaccination (Talwar, 1985; Oonk et al., 1998).
GnRH is produced in cell bodies in the hypothalamic neurons and is transported with blood
flow to its target cells in the anterior pituitary. There, GnRH binds to the gonatotrope cells
and LH and follicle stimulating hormone (FSH) are synthesized and released (Figure 7).
During the short transport from the hypothalamus to the pituitary, the hormone can be
attacked by antibodies. Binding to antibodies neutralizes GnRH either by preventing it from
diffusion through the capillary walls (due to the size of the complex) or by masking the
receptor
binding
site
on
the
GnRH
molecule
itself
(Thompson,
2000).
The
immunoneutralization results in a blockade of the GnRH-LH axis and of the synthesis of
anabolic steroids and androstenone in the testicular Leydig cells. Consequently, the taint
levels are decreased but the anabolic potential is lost as well. Today, the on-farm application
of immunization to avoid surgical castration is of primary interest as a method to inhibit
sexual development and boar taint (Caraty and Bonneau, 1986; Bonneau et al., 1994).
21
Introduction
GnRH producing neurons
eminentia mediana
primary plexus
hypophyseal portal vein blood vessels
gonadotrophe cell bodies
secondary plexus
neurohypophysis
adenohypophysis
Figure 7:
Neuro-endocrine
structures
relevant
for
“immunological
castration”
(Rosenzweig et al., 1996)
Falvo et al. (1986) vaccinated boars twice with GnRH conjugated to human serum globulin in
two different adjuvants. The second (so-called anamnestic) vaccination resulted in high
antibody titres and thus had a castration effect. LH and testosterone concentrations were
significantly reduced and weights of testes and accessory glands were lower compared to
control boars. Further, the incidence of boar taint in immunized male pigs was lower than in
intact boars. Other studies, which compared immunized boars with surgically castrated and
intact ones, confirmed that two injections are required to achieve high and thus effective
antibody titres (Bonneau et al, 1994; Manns and Robbins, 1997; Hennessy et al., 1997;
Dunshea et al., 2001). In addition, these studies found that immunized boars had less feed
intake and better feed efficiency rates compared to surgical castrates. The thickness of
backfat was significantly lower in immunized boars: Boars had 18.3 mm of backfat,
immunized boars 24.5 mm, and barrows 28.0 mm (Manns and Robbins, 1997).
One commercially available vaccine is Improvac® (Pfizer Animal Health). Improvac®
contains a modified form of GnRH in an aqueous adjuvant that causes little tissue
aggravation (Dunshea et al., 2001). All pigs in this study that were treated with two doses of
Improvac® showed an immune response, and the majority of immunized boars had no tissue
reactions following the vaccination. Testosterone levels were reduced and testicular growth
had ceased within two weeks after the second dose. 24% of the control boars had
22
Introduction
androstenone concentrations of 0.5-1.0 µg/g fat, and another 49% had levels higher than 1.0
µg/g fat. The immunized male pigs did not differ from surgical castrates. Only 3% of the
Improvac® treated boars had androstenone levels of 0.5-1.0 µg/g fat, the remaining
immunized boars were below 0.5 µg/g fat which is the threshold value for acceptance by
consumers. Thus, immunization with Improvac® is an effective method to inhibit the
testicular development and boar taint. A relevant factor regarding animal welfare is the
problem of fighting between boars. 30 pigs in this study exhibited fighting lesions, 26 were
untreated boars whereas only 4 were immunized boars. All other animals showed no fighting
scars. Surprisingly, the immunized boars showed greater daily gain and grew 7-32% faster in
the last weeks of the fattening time (Dunshea et al., 2001). It was concluded that this effect
was due to increased feed intake rather than a change in feed conversion rate. Nevertheless,
feed conversion rate and backfat in immunized boars and entire boars were similar and less
than in barrows. Turkstra et al. (2002) reported that the growth performance (i.e. average
daily gain and feed efficiency) in entire boars and in immunized boars which responded late
to the immunization was better than in barrows and in immunized, early responding boars.
These results indicate that the time schedule of the immunization and subsequent reactions
are of great importance.
Thus, immunized male pigs had a better fattening performance than surgical castrates even
though the anabolic hormones were very low. Therefore, other compensatory mechanisms
have to be considered. Metz and Claus (2003) found high GH concentrations in immunized
boars (3.88 ng/mL) similar to control boars (4.19 ng/mL). This maintenance of GH in
immunized boars was confirmed in another study (see 2.1), but it is not known why GH is
elevated. The possibility that immunized boars retain at least part of their anabolic potential
and thus an improved fattening performance and carcass quality is of high practical
relevance. It might help to compensate the costs of the vaccinations. Additionally, an
improved protein deposition at the expense of fat might be used to elevate the slaughter
weight and thus to compensate the costs for the piglet. It is necessary, however, to confirm
remaining fattening advantages by detailed studies that also include physiological
parameters to characterize metabolic reactions. Such studies should also characterize
endocrine reactions which are not directly related to testicular functions. Based on published
data, three different hypotheses were raised. They are given below.
23
Introduction
1.7
Theories underlying the own experimental studies
1.7.1 Interaction between the releasing hormones GnRH and GH-RH in the
hypothalamus or the pituitary
This hypothesis is based on the finding that the GH concentrations in immunized boars are
as high as in entire males (Table 4), whereas surgically castrated barrows exhibited much
lower GH concentrations. Thus GH levels in barrows were 31% lower than in immunized
boars although gonadal steroids were absent in both groups.
The mechanisms which maintain GH at a high male level are still unclear and cannot be put
down to the abolishment of testicular steroids and their possible negative feedback on GH
secretion (Breier et al., 1989). It rather may depend on an interaction between GnRH and
growth hormone releasing hormone (GH-RH) either in the hypothalamus or in the pituitary.
Infusion of GnRH increased LH and decreased GH, whereas infusion of GH-RH led to high
GH concentrations and low LH values (Claus and Weiler, 1994; Weiler, 1995).
Table 4:
GH concentrations (ng/mL) of entire boars, immunized boars and barrows
(Metz, 2003)
age (weeks)
boars
immunized boars
barrows
18
4.69
4.18
2.94
23
4.09
4.09
2.88
25
3.81
3.36
2.83
mean
4.19
3.88
2.88
Barrows are a good model to test this hypothesis of a possible interaction between these
releasing hormones. It is known that barrows have high concentrations of GnRH and LH due
to the surgical castration causing the lack of negative feedback of testosterone from the
testes. If there is an interaction between GnRH and GH-RH, the immunization of barrows
against GnRH (see 2.2) should lead to a drop of GnRH which in turn should result in higher
GH-RH values and consequently in higher GH concentrations and a better growth
performance (Figure 8).
24
barrows:
GnRH
GH-RH
immunized
barrows:
GnRH
GH-RH
Figure 8:
Hypothetical interaction between GnRH and GH-RH in barrows and
immunized barrows
1.7.2 Influence of different feed intake on GH and IGF-I levels
Growth and thus protein synthesis is regulated by GH and mainly by its growth factor IGF-I.
GH binds to its receptors in the liver and then stimulates the synthesis and release of IGF-I
into blood. But in addition, the GH-IGF-I axis is influenced by oestrogens and by
carbohydrates with a high glycaemic index such as glucose or starch (Figure 9).
In the pig, oestrogens lead to an additional rise in IGF-I concentrations (Claus et al., 1992;
Claus and Weiler, 1994) by increasing the expression of the GH receptors in the liver
(Gabrielsson et al., 1995). Similar to oestrogens, carbohydrates stimulate the secretion of
insulin which in turn also increases the expression of GH receptors in the liver (Brameld et
al., 1999).
The immunization against GnRH leads to a drop of testicular steroids. In consequence, after
the second vaccination oestrogens are absent, and their influence on IGF-I is no longer
prominent. All boars in our study (see 2.3) were immunized, and the two groups were fed
with different amounts of feed (2 kg vs. 3 kg). After the second vaccination the only
difference between groups was the energy supply through the diet. For this reason, we could
investigate the influence of energy on IGF-I concentrations.
oestogens
GH
liver
IGF-I
glucose
Figure 9:
25
GH-IGF-I system and regulative parameters (Claus, 2005)
Introduction
1.7.3 Imprinting of a male specific GH secretion pattern
The theory for this study (see 2.4) is based on studies in rats where perinatal presence of
testicular steroids imprints a male pattern of GH later in life (Jansson et al., 1985). This
typical male secretion was characterized by a high pulse frequency and a low baseline. In
mice and rats, sexual differentiation is not completely finished during the short gestation
period, but differentiation is extended to some short time after birth. Male pigs produce
testosterone just like rodents about 4 weeks after birth (Schwarzenberger et al., 1993). This
rise in testosterone concentration may influence GH secretion. But this rise in testosterone
cannot occur in surgical castrates because the testes are removed within the first week of life
(early castration, Figure 10). In immunized boars, in contrast, this neonatal steroid production
can take place due to the fact that the testes are still there and the immunization effect is not
relevant before the second vacciantion.
We hypothesize that the difference in GH concentrations between immunized boars and
barrows is caused by an imprinting mechanism of the neonatal steroid synthesis. Therefore,
we compared barrows which were either surgically castrated before (“early”) or after (“late”)
the neonatal rise of testosterone (Figure10).
testes function
pubertal spurt
neonatal rise
early
late
castration
Figure 10:
time course
Early and late castration in the time course of pubertal development of boar
testes function
26
Experimental Studies
2
Own Experimental Studies
2.1
Short-term endocrine and metabolic reactions before and after second
immunization against GnRH in boars
(R Claus, M Lacorn, K Danowski, MC Pearce, A Bauer
Vaccine 2007, 25: 4689-4696)
Article included in consent of Elsevier.
28
Vaccine 25 (2007) 4689–4696
Short-term endocrine and metabolic reactions before and after
second immunization against GnRH in boars
Rolf Claus a,∗ , Markus Lacorn a , Katrin Danowski a , Michael C. Pearce b , Aneka Bauer a
a
Universität Hohenheim, Institut für Tierhaltung und Tierzüchtung, Garbenstraße 17, 70599 Stuttgart, Germany
b Veterinary Medicine Research and Development, Pfizer Ltd, Ramsgate Rd, Sandwich, Kent CT13 9NJ, UK
Received 4 December 2006; received in revised form 27 February 2007; accepted 2 April 2007
Available online 20 April 2007
Abstract
Immunization of boars against GnRH inhibits synthesis of testicular steroids including androstenone (sex odour). Timing of the second
vaccination (anamnestic reaction) should occur as late as possible to maintain anabolic effects of testicular hormones, but early enough to
remove androstenone from body fat. Five catheterized boars received the second dose (Improvac® ) at age 22 weeks. Titre, hormones and
parameters reflecting protein turnover were determined in blood. An increased antibody titre and drop of LH and steroids occurred within 5
days. Metabolism adapted after 7 days. Results from this study in conjunction with previous work suggest that after two doses of Improvac
given 4 weeks apart, clearance of androstenone from body fat may be achieved as early as 3 weeks after the second vaccination. Thus, it might
be possible to extend the duration of anabolic effect in male pig production.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: Immunological castration; Boar sex odour; Anamnestic vaccination
1. Introduction
Due to the anabolic function of their testicular hormones,
boars have superior fattening performance and carcass composition compared with barrows, while gilts are intermediate
in these traits [e.g. 1–4]. This hierarchy is explained by
the availability of gonadal hormones. Gilts secrete estrogens
from the ovary, whereas testicular steroid synthesis in boars
includes androgens and high amounts of estrogens [5] which
differ in their anabolic mechanisms. Androgens improve protein synthesis via muscular receptors [6] and decrease protein
turnover, mainly by interacting with the glucocorticoid receptor [7]. In contrast, estrogens act indirectly by stimulating
growth hormone (GH) secretion from the pituitary gland
and, even more pronounced, the GH-dependent secretion of
the insulin-like growth factor 1 (IGF-1) from the liver by
increasing the hepatic GH-receptors [8].
∗
Corresponding author. Tel.: +49 459 22455; fax: +49 459 22498.
E-mail address: [email protected] (R. Claus).
0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2007.04.009
In addition to sex hormones, boars also synthesize
androstenone in the testes. This steroid has no hormonal
activity [9] but functions as a pheromone [10]. Androstenone
accumulates in adipose tissue and its urine-like smell is
regarded as a major cause of boar taint [11] which may result
in rejection of meat from uncastrated pigs by a considerable
proportion of consumers [12]. Manifold attempts to suppress
androstenone but maintain anabolic status have been unsuccessful due to the common biosynthesis of anabolic steroids
and androstenone in the testes. Implantation of anabolic sex
hormones was effective to decrease steroid synthesis due
to their negative feedback on gonadotropins. The anabolic
effect cannot be used, however, due to residue considerations [13]. Surgical castration without anaesthesia has usually
been used to control boar taint but is an important animal welfare issue which led to legal intervention e.g. in
Norway. Immunological castration by immunization against
gonadotropin releasing hormone (GnRH) [e.g. 14–16] is
increasingly discussed as an alternative to surgical castration.
However, immunization blocks the GnRH-luteinizing hormone (LH)-axis and thus also synthesis of anabolic steroids
4690
R. Claus et al. / Vaccine 25 (2007) 4689–4696
in the Leydig cells. In consequence, immunization leads not
only to inhibition of boar taint but also anabolic effects. So
far, the only commercially available vaccine for immunological castration is Improvac® [17] but it is not registered in
all countries. The manufacturer recommends that two doses
are given at least 4 weeks apart with the second dose given
4–6 weeks prior to slaughter. An anamnestic immunological
response occurs resulting in high levels of antibody against
GnRH 10–14 days after the second dose, as described by the
manufacturer. Such a scheme not only suppresses boar taint,
but also retains anabolic effects for a large part of the fattening
period which probably explains the excellent performance
data in immunized boars found in a field study [18]. In a previous study, we used an early onset of immunization: the first
dose was given at age 10 weeks followed by two additional
doses at age 16 and 23 weeks to abolish anabolic steroids over
most of the fattening period. We found not only inhibition of
androstenone but also a loss of anabolic function. Nitrogen
retention in this study did not differ between immunocastrates and barrows but was about 24% higher in boars [19].
Therefore, the timing of vaccination in the fattening period
is of high practical relevance.
To optimize the vaccination schedule, the following need
to be considered: time till sufficient antibody formation after
the second dose of vaccine; time to cessation of Leydig cell
function and the resulting drop in peripheral steroid concentrations; time required for clearance of androstenone stored in
adipose tissue. Only the latter question had been answered by
earlier published studies: androstenone was completely eliminated from slaughterweight boars approximately 2 weeks
after castration [20]. However, clearance from adipose tissue
is age dependent, and it may take more than 6 weeks in older
boars [20].
Detailed data on the immunological and metabolic
responses to immunological castration are still missing.
The aim of this study was to examine immunological, hormonal, and metabolic responses following the second dose
of Improvac® in a two-dose immunocastration schedule.
2. Materials and methods
2.1. Animals
Five German Landrace boars were obtained from the University of Hohenheim swine herd and kept in individual
pens (3 × 2.5 m). For blood collection, each boar was fitted
with a cephalic vein catheter as described in detail earlier
[21]. Catheters were surgically implanted at an age of 17
weeks (body weight range: 52–61 kg) under general anesthesia. Average duration of anesthesia did not exceed 30 min and
pigs were fully recovered from surgery 2 days later. Animals
were then immunized at weeks 18 and 22 with 2 mL doses
of Improvac® subcutaneously in accordance to the manufacturer’s recommendations (CSL Ltd., Parkville, Australia).
Deuterium oxide administrations for later determination of
Table 1
Feed composition
Component
Wet weight (g/kg)
Barley
Triticale
Wheat
Pea
Soybean meal
Vitamin and mineral premix
Calcium carbonate
Soybean oil
373
200
170
70
150
25
2
10
total body fat (see below) were performed in ages 20 and
25 weeks. At age 28 weeks, the boars were slaughtered. The
pigs were fed twice daily at 8:00 and 15:00 h with portions of
1.5 kg of a ration (Table 1) containing 16.7% crude protein
and 13.5 MJ ME/kg.
Blood was sampled daily before feeding at 8:00 h into heparinized vials and plasma was stored at −20 ◦ C until assayed.
Catheters were rinsed using sterile heparinized saline after
each sampling. The whole experiment including the cannulation had been approved by the local animal welfare committee
of the state of Baden-Württemberg.
2.2. Analytical methods
2.2.1. GnRH-antibody titre
For titre determination, GnRH (Bachem, Weil am Rhein,
Germany) was dissolved in PBS and diluted in 0.1 M carbonate buffer (pH 9.6) at a concentration of 10 ng/mL. One
hundred ␮L/well were pipetted into Immobilizer Amino
microtiterplates (Nunc, Wiesbaden, Germany) and allowed to
react for 1 h at 37 ◦ C under shaking. Plates were emptied and
blocked with testing buffer (0.12 M NaCl, 0.02 M Na2 HPO4 ,
0.01 M EDTA, 0.1% hydrolyzed gelatine, 0.05% Tween 20,
0.002% phenol red, 0.005% chlorhexidine gluconate; pH 7.2)
overnight at 4 ◦ C. All subsequent washing steps were performed using 10% PBS containing 0.05% Tween 20. Plasma
samples from boars were diluted either 1:1600 or 1:3200
with testing buffer and 100 ␮L of each dilution per well was
incubated for 2 h at 37 ◦ C. For the next step, sheep-anti-pig
IgG was biotinylated using EZ-link-NHS-LC-Biotin according to the manufacturer’s instruction (Pierce Biotechnology,
Rockford, IL, USA) and added at 3.2 ng/well. StreptABComplex (Dako, Hamburg, Germany) was added at a dilution of
1:5000 in testing buffer and incubated for 30 min at 37 ◦ C in
the dark. For colour reaction tetramethylbenzidine/H2 O2 in
acetate buffer was used. Reaction was stopped after 40 min
at 37 ◦ C using 2 M H2 SO4 . Optical density (OD) was determined at 450/690 nm and used to characterize the titre
quantitatively.
2.2.2. LH
Radio-immunological determination of LH was performed as described earlier [22]. Because there was no need to
characterize LH pulsatility, only daily samples were used for
determination of LH. The intra- and interassay coefficients of
variation were 10% and 8.3%, respectively, at a concentration
of 0.6 ng/mL. The lower limit of detection was 0.02 ng/mL.
2.2.3. Testosterone
The radio-immunoassay used for measurement was
described earlier [23] and revealed a coefficient of variation
between days of 6.8% (2.95 ng/mL). The lower limit of detection was 0.05 ng/mL and average recovery was above 95%
for concentrations ranging from 0.5 to 10 ng/mL.
2.2.4. Androstenone
For determination of 5␣-androst-16-en-3-one (androstenone) 1 mL plasma was extracted with 3 mL n-hexane by
shaking in a sealed glass vial for 30 min. After freezing at
−30 ◦ C for 30 min, the liquid hexane phase was decanted in
a glass vial and evaporated in a rotary evaporator for 10 min
until only a small volume of hexane was left. The vial was vortexed and evaporated to complete dryness within 5 min in the
evaporator. We found earlier that such a short period does not
lead to losses of androstenone. Dried residues were resolved
in 100 ␮L methanol containing 5 ng of 5␣-androstan-3-one
as an internal standard for the gas chromatographic-mass
spectrometric (GC-MS) determination. The system comprised a GC-17A coupled to a QP 5050 mass spectrometer
(Shimadzu, Duisburg, Germany). GC-MS conditions were
as follows: column, 10 m × 0.25 mm Zebron Amino Acid
(Phenomenex, Aschaffenburg, Germany); injection volume,
3 ␮L; injection port temperature program, 110 ◦ C (1 min),
300 ◦ C (250 ◦ C/min); carrier gas was Helium, 3 mL/min (split
less); column temperature program, 80 ◦ C (2 min), 300 ◦ C
(30 ◦ C/min), 300 ◦ C (3 min); transfer nozzle temperature,
280 ◦ C; multiplier voltage, 1.75 kV; single ion monitoring
at m/z 272 and 274 (m/z 257 as another characteristic ion
for androstenone could not be used because of co eluting substances). For calibration androstenone-free plasma of
a barrow was spiked with 0.5, 1.0, 2.5, 5, and 10 ng/mL.
Repeatability was determined by measurement of a boar
plasma sample either on the same day (n = 5, intraassay coefficient of variation of 4.9%) or on consecutive days (interassay
coefficient of variation of 8.2%). Sensitivity (threefold signalto-noise ratio) was evaluated by spiking plasma of a barrow
with increasing amounts of androstenone and revealed a
detection limit of 0.3 ng/mL.
To save limited plasma, only samples of every fifth
day during the whole experiment were measured for
androstenone, except samples taken 2–8 days after the second
dose of Improvac® to display short term daily changes.
2.2.5. IGF-1
Concentrations of IGF-1 in plasma were determined as
described before [24]. In brief, samples were extracted with
HCl/ethanol and measured using a specific double-antibody
RIA. Coefficients of variation within and between assays
were 8.4% (160 ng/mL) and 5.7% (183 ng/mL), respectively.
4691
2.2.6. Urea
Nitrogen excretion is mainly represented by urea but transfer of pigs into metabolic crates to obtain urine and faeces is
stressful, resulting in metabolic artefacts. Instead, urea was
measured in plasma using microtiterplates because it had
been shown previously, that urea determination is an excellent analytical parameter to characterize N-retention [25]. In
brief, plasma was diluted four-fold and pre-incubated with
glutamic-dehydrogenase to remove ammonia by reaction
with oxoglutarate, ADP and NADH. After addition of urease
released ammonium was quantified at 340 nm using urea standards. Intraassay and interassay coefficients of variation were
8.9% (n = 10; 332 ␮g/mL) and 9.7% (n = 10; 332 ␮g/mL),
respectively.
2.2.7. Total body fat
Composition of the fat-free body mass is strongly correlated with the total body mass and not influenced by the body
fat content [26]. Fat-free body mass is calculated from total
body water which is determined by application of a known
dose of D2 O and measurement of D2 O content in body water
after equilibrium. The difference between total body mass
and fat-free body mass is the fat content. For determination,
animals were weighed and injected intravenously through the
catheter with D2 O (70%; Chemotrade, Leipzig, Germany) at
0.21 g 70% D2 O/kg body weight. The exact amount given
to each pig was determined by weighing the filled syringe
before and after injection. Following administration of D2 O
the catheter was rinsed with 40 mL sterile heparinized saline.
After equilibration with body water, three 20 mL blood samples were drawn at 4, 4.5, and 5 h after D2 O application
into heparinized vials and stored at −20 ◦ C. For clean-up,
the method described by Tissier et al. [27] was used. Five
millilitres of the blood were freeze-dried and the water phase
sublimated in a cryo trap using liquid nitrogen. D2 O-content
was determined at 3960 nm in an infrared spectrophotometer
using D2 O calibrators (0, 252, 311, 505 mg/kg). Intraassay
and interassay coefficients of variation were 0.2% (n = 5)
and 2.2% (n = 10), respectively. Total body fat content was
calculated as described by Susenbeth [26].
2.2.8. Hydroxyproline (OH-Pro)
Hydroxyproline in blood plasma and urine reflects collagen breakdown. This breakdown is lowered when protein
synthesis and deposition is reduced as well [25,28]. Lopes et
al. [25] also described the method used in this study. In brief,
400 ␮L 7.5 M HCl was placed in a screw-capped vial and
100 ␮L plasma added to the acid. Hydrolysis was performed
for 12 h at 110 ◦ C. The hydrolysates were evaporated in a
heating chamber and redissolved in 5 mL bidistilled water.
Derivatization and HPLC-analysis were modified according
to Einarsson [29]. In brief, cis-hydroxyproline was added
before derivatization as an internal standard and primary
amino acids are derivatized using 9-fluorenylchloroformiate.
After extraction with diethyl ether, secondary amino acids
were derivatized with o-phthaldialdehyde and diluted 20-
4692
fold. Chromatographic conditions were as follows: column,
Lichrospher 100 RP 18 125 mm × 4.6 mm 5 ␮m (pre-column
of the same material); injection volume, 10 ␮L; eluent A:
0.1% aqueous trifluoroacetic acid/acetonitrile (70:30, v:v),
eluent B: acetonitrile; gradient elution (minutes/% eluent
B): 0/0, 2/0, 8/40, 8.5/100, 11.5/100, 12/0, 15/0, flow,
1.5 mL/min; fluorescence detector, ex/em: 255 nm/325 nm.
Calibration was performed with OH-Pro standards. Coefficients of variation within and between assays were 2.7%
(22.6 ␮g/mL) and 8.5% (21.8 ␮g/mL), respectively. To save
limited plasma, only samples of every fifth day during the
whole experiment were measured.
2.3. Statistics
Data are presented as daily means ± S.E.M. Day 0 is the
day of the first immunization and day 28 the day for second
immunization. For statistical evaluation the mean for period
I (days 0–28) and period II (day 39 until slaughter on day
72) were calculated for every animal and tested for differences between these periods using paired t-test. The period
between days 29 and 38 are omitted because of increasing or
decreasing values. To determine the onset of antibody titre
development and rise of urea in each pig, the mean value
and the standard deviation (S.D.) were calculated from day
0 until day 28 (second immunization). The first value above
the mean value plus the threefold S.D. (p < 0.05) was defined
as the onset. Similarly, LH, testosterone, androstenone, IGF1, and OH-Pro were checked for a decrease. In this case, the
mean value and the standard deviation (S.D.) were calculated
from the last 4 weeks of the experiment (days 44–72). For
analysis of significant trends in titre development between
days 37 and 72 Neumann’s test was used [30]. Correlations
between parameters were determined by Spearman’s rank
correlation coefficient because LH determination led to concentrations below the detection limit. The Statistical Package
for the Social Sciences (SPSS Version 12.0; Chicago, IL,
USA) was used.
Fig. 1. Development of GnRH-antibody titres for each pig (individual antiserum dilution given in brackets). First Improvac immunization was on day
0, whereas the second immunization was on day 28.
Changes in the concentrations of LH are shown in Fig. 2.
They decreased from mean values of 0.158 ng/mL to concentrations of 0.03 ng/mL which is close to the detection limit.
This significant decrease (p < 0.01) was found within 4–8
days after second dose. Therefore, there was no apparent
time lag between antibody development and LH decrease.
None of the pigs showed an increase in LH during the whole
period after the second vaccination, so that the low antibody
titres found in three of the pigs at the end of the sampling
period were still sufficient to suppress GnRH and LH in
consequence.
Similar to LH, testosterone (Fig. 3) remained at high mean
concentrations of about 2.75 ng/mL after the first immunization and decreased to values of about 0.25 ng/mL (p < 0.001)
3. Results
Developments of antibody titres against GnRH are shown
in Fig. 1. It was found that all pigs had a significant increase in
antibodies against GnRH from the period before the second
immunization and thereafter, but the extent of antibody formation differed between individuals. While pigs 1, 4, and 5
revealed high antibody titres with an extinction of more than
2, pigs 2 and 3 exhibited a weak titre development which had
been measured at a dilution of 1:1600 to obtain extinctions
above 1. Titres raised within 3–5 days after second dose in
all pigs, reaching the highest levels between days 4 and 6.
Thereafter titres decreased (p < 0.05, Neumann Test), except
in one pig which had a second peak 4 weeks after the booster.
At the end of the sampling period all pigs still had elevated
titres but they were about half the peak values.
Fig. 2. LH in plasma (n = 5; daily means ± S.E.M.). First immunization was
on day 0, whereas second immunization was on day 28 (dotted line).
Fig. 3. Testosterone in plasma (n = 5; daily means ± S.E.M.). First immunization was on day 0, whereas second immunization was on day 28 (dotted
line).
5–10 days after second dose. No rise of testosterone was
found before the end of sampling.
Before second dose the androstenone concentrations differed between individuals (range of mean values from days
0 to 27: 0.62–2.67 ng/mL). The development of plasma
androstenone, concentrations is shown in Fig. 4. Mean values
decreased significantly (p < 0.05) from 1.48 to 0.34 ng/mL
after the second dose. Basal concentrations in an order of
0.35 ng/mL were reached 4–8 days after second dose. As for
testosterone, there was no rise of concentrations until the end
of the experiment.
Fig. 4. Androstenone in plasma (n = 5; daily means ± S.E.M.). First immunization was on day 0, whereas second immunization was on day 28 (dotted
line).
4693
Fig. 5. IGF-1 in plasma (n = 5; daily means ± S.E.M.). First immunization
was on day 0 whereas second immunization was on day 28 (dotted line).
Measurement of IGF-1 in plasma (Fig. 5) revealed a
decrease of about 36% from mean values of 186 to 119 ng/mL
(p < 0.01). The decrease started 5 days after second vaccination and reached basal values, 6–10 days after the booster.
Decreased N-retention due to the drop of anabolic hormones is reflected by the concentrations of urea in plasma
(Fig. 6). Urea also showed an immediate reaction after second
dose (203 ␮g/mL) which was significantly higher (p < 0.001)
compared to the level before the booster (126 ␮g/mL). In
contrast to urea, measurement of OH-Pro (Fig. 7) revealed a
decrease of about 24% from mean values of 26.9–20.4 ␮g/mL
(p < 0.01). Basal values were reached 6–10 days after sec-
Fig. 6. Urea in plasma (n = 5; daily means ± S.E.M.). First immunization
was on day 0 whereas second immunization was on day 28 (dotted line).
4694
Table 2
Correlation between parameters investigated (** p < 0.001)
IGF-1
Urea
LH
Testosterone
GnRH-Titre
Androstenone
IGF-1
Urea
–
−0.455**
–
LH
Testosterone
0.398**
0.455**
−0.563**
–
−0.634**
0.526**
–
Fig. 7. Hydroxyproline in plasma (n = 5; daily means ± S.E.M.). First immunization was on day 0, whereas second immunization was on day 28 (dotted
line).
ond immunization. The total body fat content after the first
and second immunization revealed slightly increasing values
from 19.5% (week 20) to 21.8% (week 25), which was not
significant.
Correlations between the parameters determined are summarized in Table 2. Plasma androstenone and OH-Pro were
not correlated significantly. All other parameters, either representing an anabolic metabolism or being members of the
hormonal regulators of anabolism, were significantly correlated (p < 0.001).
4. Discussion
Many field studies have demonstrated the efficacy of
GnRH-immunization using different antigens, including
Improvac® [15–18,31]. A vaccination scheme with two doses
is generally recommended. The first vaccination is followed
by a primary immune response in which a limited amount
of antibody is produced with a relatively high proportion
of non-specific IgM antibodies, and immunological memory
is established. Following the second dose, there is a rapid,
large and persistent increase in titres of circulating antibodies dominated by high affinity IgG [32]. In contrast to our
study in which the first immunization had no obvious effect,
GnRH-Titre
−0.371**
0.603**
−0.745**
−0.501**
–
Androstenone
0.466**
−0.489**
0.580**
0.669**
−0.744**
–
OH-Pro
0.485**
−0.575**
0.534**
0.470**
−0.525**
0.367
other studies detected biological effects after a single injection, probably due to the use of more aggressive adjuvants
[33–35].
To date detailed temporal relationships between the second dose and endocrine and metabolic reaction have not
been examined in detail. Our data from cannulated pigs
revealed a very rapid increase of antibody formation after
the second dose. Quantitative aspects, however, were highly
variable depending on the individual. Nevertheless, it was
fully sufficient to release the castration effects. The continuous decrease after the maximal titre might allow recovery
of testicular function before slaughter if the second dose of
a vaccine is given too early. Timing of the second dose of
Improvac® should be optimised to achieve a high enough titre
of antibodies to ensure the castration effect in all boars and,
secondly, make optimal use of the anabolic effect of gonadal
hormones when they have reached levels typical in the mature
boar. Currently, the manufacturer recommends giving the
dose 4–6 weeks before slaughter. One study using a limited
number of boars reported that in slaughter weight boars, 3
weeks are sufficient for androstenone to be eliminated from
adipose tissue after surgical castration [20]. Further research
is needed to determine whether the currently recommended
4–6 weeks window before slaughter can be shortened and
still achieve reliable boar taint suppression but an extended
period of anabolic function.
Age-dependent testicular function is well investigated in
the boar. Apart from a transient rise of testicular endocrine
function 2–4 weeks postnatally [36], the pubertal spurt starts
at around 16–17 weeks leading to peak levels of steroids in
blood plasma at an age of about 19 weeks [37]. Thereafter,
they decrease slightly and stabilize at a high level [37–39].
As shown in the present study, LH and testosterone concentrations decreased immediately with the rise of titre. The
rapid decrease of testosterone is explained by the effect of
LH on cholesterol side chain cleavage which is the rate limiting step in testicular steroidogenesis [40,41]. Maintenance
of the titre of antibodies against GnRH and thus withdrawal
of LH for prolonged periods led to Leydig cell involution
characterized by an almost complete loss of cytoplasm [22].
The gradual decrease of testosterone is not a reflection of low
levels of ongoing synthesis, but can be explained by the clearance of testosterone which was stored in adipose tissues of the
boar [42] and becomes measurable in blood plasma. The same
kinetic can be assumed for androstenone, but androstenone
is more lipophilic so it achieves much higher tissue concen-
trations in adipose tissue and release from the tissue is more
protracted [43].
In contrast to gonadal steroids, the decrease of IGF-1 was
much slower. IGF-1 is increased by estrogens [44]. Estrogens
were not measured in this study due to limited availability
of blood plasma but have been shown earlier to be highly
correlated to androgens [43] and to decrease because of
GnRH-immunization [19]. The slow decrease of IGF-1 after
second dose is explained by its long half life, which is due to
binding to specific binding proteins [45].
The loss of anticatabolic androgens and the decrease
of the estrogen/IGF-1-dependent proliferative functions are
also reflected by the metabolic parameters. The increase of
urea signals enhanced protein degradation and N-excretion.
In contrast to urea, OH-Pro decreased after second vaccination. Synthesis and breakdown of collagen reveal a
sex-dependent equilibrium and it was shown that androgens
primarily stimulate collagen synthesis [46,47]. The reduced
OH-Pro concentrations thus reflect a decrease of collagen
breakdown to compensate for the drop in collagen de novo
synthesis. Apart from the rapid drop of protein anabolism
after the second vaccination, it is also known that the voluntary feed intake increases remarkably due to the absence of
both estrogens and androgens [48]. It is advisable therefore
to adapt amount of daily feed supply and feed composition around the time of second vaccination to finishing
rations for barrows, to avoid excessive fat accumulation in the
carcass.
Acknowledgements
We thank Dr. U. Weiler who participated in animal surgery
and C. Fischinger, B. Dunne, and M. Mecellem for care of
the animals. A. Wagner, S. Knöllinger and S. Mayer performed excellent laboratory work and B. Deininger prepared
the manuscript.
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2.2
Effects of immunization against GnRH on gonadotropins, the GH-IGF-I-axis and
metabolic parameters in barrows
(A Bauer, M Lacorn, K Danowski, R Claus
Animal 2008, 2: 1215-1222)
Article included in consent of Cambridge University Press.
38
animal
Animal (2008), 2:8, pp 1215–1222 & The Animal Consortium 2008
doi:10.1017/S1751731108002346
Effects of immunization against GnRH on gonadotropins,
the GH-IGF-I-axis and metabolic parameters in barrows
A. Bauer, M. Lacorn, K. Danowski and R. ClausInstitut für Tierhaltung und Tierzüchtung, Universität Hohenheim, Garbenstr. 17, 70599 Stuttgart, Germany
(Received 2 October 2007; Accepted 26 March 2008)
Surgically castrated male piglets (barrows) reveal an increase in LH and a decrease in GH compared to untreated boars. Boars
that were castrated by immunization against gonadotropin releasing hormone (GnRH) have decreased LH but maintain GH. The
difference in GH levels between barrows and immunological castrated boars cannot be explained by testicular steroids because
they are low in surgical and immunocastrated boars as well. Therefore, differences in GH concentrations might be due to an
interaction between GnRH and growth hormone releasing hormone (GRH) in the hypothalamus or the pituitary. This hypothesis
was tested with twelve male piglets that had been castrated within 1 week postnatally and fitted with indwelling cephalic vein
catheters at 17 weeks of age. They were split into a control group and an immunized group (each n 5 6). Vaccination with
R
was performed at 18 and 22 weeks of age. Specific radioimmunoassays were used for hormone determinations
Improvac
(GH, LH, FSH, testosterone and IGF-I). Additionally, metabolic responses were evaluated by measuring analytical parameters
that characterize protein synthesis and breakdown, and body fat content. The second vaccination led to a rapid decrease of LH
below the limit of detection whereas FSH decreased more slowly, over a period of 5 weeks, from 2.2 to 0.5 ng/ml. This level
of FSH, which corresponds to boar-specific concentrations, was maintained thereafter. GH decreased with increasing age but
was not influenced by vaccination and remained at a low concentration typical for barrows. Similarly, IGF-I was not altered
by vaccination. Consequently, metabolic status was not changed by immunization. It is concluded that the difference in GH
levels between surgical and immunocastrated boars is not explained by an interaction between GnRH and GRH.
Keywords: pig, GnRH immunization, metabolism, GH, FSH
Introduction
Boars synthesize high amounts of androgens, oestrogens
and the pheromone 5a-androst-16-en-3-one (androstenone) in testicular Leydig cells (Claus and Hoffmann, 1980).
Apart from their regulatory role in reproductive functions,
androgens and oestrogens have a high anabolic potential.
Androstenone has no hormonal activity but stimulates
female reproductive functions as a pheromone (Reed et al.,
1974) and is mainly responsible for the unpleasant ‘boar
taint’ (Patterson, 1968). To prevent this taint, male piglets
are usually castrated surgically without anaesthesia shortly
after birth, even though fattening performance, nitrogen
retention and the ratio of lean to fat are inferior in barrows
compared with boars. In recent years, castration without
anaesthesia has become an important animal-welfare issue.
Norway expects to ban surgical castration completely
in 2009.
-
E-mail: [email protected]
Currently, there are two approaches under discussion for
the control of boar taint, which will not compromise animal
welfare. The first is surgical castration with anaesthesia
(Prunier and Bonneau, 2006; Thun et al., 2006; Zöls et al.,
2006). The second is the use of vaccines (immunological
castration) to inactivate gonadotropin releasing hormone
(GnRH), causing a drop of LH, a reduction of steroid
synthesis and, consequently, decreased taint levels. FSH is
reported to remain unchanged after immunization, probably
due to a different regulation via the activin system (Li et al.,
1998). Metz and Claus (2003) found that GH concentrations
in immunocastrated boars remained at the same high level
typically found in intact males. Normal barrows, however,
have GH levels that are 31% lower than immunocastrates
(Metz and Claus, 2003) although steroid concentrations are
low as in barrows. Thus, the mechanism that maintains GH
in immunocastrates at a level typical for intact boars cannot
be due to the feedback effect of steroids on GH secretion
(Breier et al., 1989). Rather, it may depend on an interaction
between GnRH and growth hormone releasing hormone
1215
Bauer, Lacorn, Danowski and Claus
(GRH) either in the hypothalamus or in the pituitary. In the
pituitary, such an interaction may occur by cells that are
thought to be able to synthesize GH and gonadotropins
(Childs, 2000; Childs et al., 2000). The hypothesis is substantiated by the infusion of GnRH in pigs, which led to an
increase of LH by 86% but to a concomitant decrease of GH by
18%. Infusion of GRH in turn elevated GH by 78% and
decreased LH by 16% (Claus and Weiler, 1994). Since immunization against GnRH leads to an inactivation of GnRH, a rise of
GH might be expected. Barrows are an appropriate model to
clarify this hypothesis because they have higher levels of GnRH
and gonadotropins due to the absence of negative feedback
by gonadal steroids compared to boars. Immunization against
GnRH should then lead to a rise of GRH and GH. Because GH
is known as the anabolic ‘master-hormone’, such a rise should
also be reflected by corresponding changes of parameters that
characterize an anabolic metabolism. Such a mechanism might
also be of practical relevance to improve the growth performance of barrows.
Materials and methods
Study design
Experimental pigs were German Landrace barrows that had
been surgically castrated at 1 week of age. The aim of the
study was to compare groups of six immunized and six nonimmunized barrows. Because the maintenance of catheters
and animal welfare over prolonged periods require extensive
care, it is not possible to keep 12 cannulated pigs at the same
time. Consequently, the study was performed in two replicates where each replicate comprised three non-immunized
barrows as controls and three immunized barrows. Care was
taken to provide the same environment in the two replicates.
For that reason, animals were kept under a constant light
regime (12 h light : 12 h dark). Each pig was kept separately in
an individual pen measuring 3 3 2.5 m, and fed twice daily at
0800 and 1500 h with 1.5 kg of a diet containing 16.7%
crude protein and 13.5 MJ ME/kg (Table 1). The barrows were
slaughtered at 28 weeks of age.
Immunization
Immunization was performed with GnRH, which had been
linked to a glycoprotein carrier. This antigen is commercially
R
available (Improvac
, Pfizer Animal Health, Parkville,
Australia) and was administered in accordance with the
manufacturer’ recommendations. The first 2 ml dose was
given when pigs were 18 weeks old; a second 2 ml dose
was given 4 weeks later at 22 weeks of age.
The control group was not injected because the adjuvant
alone is not available. Furthermore, it is known that it does
not lead to tissue reactions (Dunshea et al., 2001).
Blood sampling
For blood sampling, each barrow was fitted with a cephalic
vein catheter under general anaesthesia at 17 weeks of age
(BW range: 50 to 57 kg in replicate 1; 53 to 58 kg in
replicate 2) as described earlier (Claus et al., 1990). Blood
1216
Table 1 Feed composition
Component
Barley
Triticale
Wheat
Pea
Soybean meal
Calcium carbonate
Soybean oil
Wet weight (g/kg)
373
200
170
70
150
25
2
10
samples were drawn daily at 0900 h in heparinized vials
and stored at 2208C after centrifugation.
Metabolic measurements
To determine body fat content, the deuterium oxide method
was performed at an age of 20 and 25 weeks as described
earlier (Claus et al., 2007). Protein synthesis was measured by
13
C-leucine infusion at the day of slaughter as described
below. For GH determination, additional samples (window
sampling) were drawn for 24 h every 20 min at 19 and 24
weeks of age. Catheters were rinsed using sterile heparinized
saline after each sampling. The whole experiment including
the cannulation had been approved by the animal welfare
committee of the state of Baden-Württemberg.
Analytical methods
GnRH–antibody titre. GnRH (Bachem, Weil am Rhein,
Germany; 15.6 ng/well in 0.05 M carbonate buffer, pH 9.6)
was coated on MaxiSorp microtitre plates (Nunc, Wiesbaden,
Germany) for 1 h at 378C under shaking. Blocking with
testing buffer (overnight at 48C) and washing steps have
been described before (Claus et al., 2007). Plasma samples
were diluted 1 : 1600 with testing buffer and 100 ml of each
dilution per well was incubated for 2 h at 378C. Thereafter,
rabbit anti-swine IgG-horseradish peroxidase (P0164, Dako,
Hamburg, Germany) was added at a dilution of 1 : 10 000 in
testing buffer and incubated for one hour at 378C in the
dark. Colour reaction and optical density determination
were measured as described previously (Claus et al., 2007).
Repeatability was determined using three different plasma
samples from immunized boars with different known titres
(n 5 7). Interassay coefficients of variation were 24%, 9.4%
and 3.6% for extinctions of 0.486, 2.754 and 3.903,
respectively.
LH. Earlier determinations of LH in barrows were based on
window sampling at 20-min intervals and led to an erratic
pattern of LH concentrations (Metz, 2003). Because the
diurnal pattern of LH was not relevant for this publication,
we preferred to determine LH in samples taken at a standardized time over prolonged periods. Such a sampling
regime was shown earlier to reflect major changes over
prolonged periods without short-term fluctuation (Claus et al.,
2007). LH in samples taken every third day during the
Immunization of barrows against GnRH
whole experiment was measured by radioimmunoassay
(RIA) (Wagner and Claus, 2004). The specific antiserum
(AFP15103194) was kindly provided by Dr Albert Parlow
(NIDDK, Torrance, CA, USA) and a highly purified pLH
standard (AFP11043B) was used for radio-iodination and
for calibration. The coefficients of variation within one day
and between days were 6.8% (mean: 0.4 ng/ml) and 6.7%
(mean: 0.37 ng/ml), respectively. The lower limit of sensitivity was 0.024 ng/ml. For determination of recovery,
spiked samples were prepared, which reflect mean physiological concentrations. Thus the recovery for 0.5 ng/ml
was 105% (n 5 6).
FSH. FSH was measured in samples from every third day
by RIA (Wagner and Claus, 2004). A specific antiserum
(AFP2062096) was provided by Dr Parlow (NIDDK, Torrance,
CA, USA) and a highly purified pFSH standard (AFP10640B)
was used for radio-iodination and for calibration. The
coefficients of variation within one day and between days
were 3.3% (mean: 1.92 ng/ml; n 5 10) and 9.2% (mean:
1.7 ng/ml; n 5 5), respectively. The lower limit of sensitivity
was 0.03 ng/ml and the recovery for spiked samples with
2.5 ng/ml was 105% (n 5 6).
Testosterone. A specific RIA including a solvent extraction
step was used as described earlier (Wagner and Claus,
2004) to measure plasma testosterone every third day.
Antiserum against testosterone-3-carboxymethyloxime-BSA
was raised in rabbits. Cross-reactivities with other steroids
were 30% for dihydrotestosterone and 0.5% for dehydroepiandrosterone, whereas 17ß-estradiol, progesterone and
epitestosterone showed cross-reactivities less than 0.1%.
Repeatability was determined by measurement of spiked
samples on consecutive days (n 5 12) and the interassay
coefficient of variation was found to be 8% for concentrations between 0.5 and 2.5 ng/ml. The lower limit of
detection was 0.05 ng/ml. The average recovery after
extraction was above 95%.
GH. Due to the pulsatile secretion pattern of GH, which is
more important than the mean level, this hormone was
determined in plasma obtained by window sampling.
Determination was performed as described before (Claus
et al., 1990). A specific antiserum (AFP422801) was kindly
provided by Dr Parlow (NIDDK, Torrance, CA, USA) and a
highly purified pGH standard (AFP10864P) was used for
radio-iodination and calibration. The intraassay coefficient
of variation was 9.3% at a concentration of 4.2 ng/ml
(n 5 10) while the interassay coefficient of variation was
10% (mean: 3 ng/ml, n 5 6). Recovery was above 95% for
spiked samples (1.5 to 6 ng/ml). Each GH profile was
evaluated for pulses. The mean for all 74 samples per
window for each animal was determined. A pulse was
assumed when at least two consecutive GH concentrations
exceeded this mean value by at least 50%. The maximal
level was the mean of the maximal concentrations of these
pulses. The base levels were calculated by the mean of the
14 lowest values. The frequency was defined as the number
of pulses per 24 h.
IGF-1. Concentrations of IGF-1 were determined by a
double-antibody RIA in daily plasma samples after HCl/
ethanol-extraction using a specific antiserum raised in
rabbits (Claus et al., 1992). The highly purified IGF-I standard
was obtained from Gro Pep (CU020, Adelaide, Australia)
and was used for radio-iodination and for calibration.
Extraction yield was between 61% and 70% for samples
spiked from 75 to 400 ng/ml (n 5 9). The coefficients of
variation within and between assays were 6.9% and 5.7%
for concentrations of 186 ng/ml (n 5 10) and 183 ng/ml
(n 5 9), respectively.
Urea. Urea in plasma was measured to characterize protein
degradation and therefore nitrogen excretion. Determination was performed on microtitre plates as reported earlier
(Claus et al., 2007). Intra- and interassay coefficients
of variation were 8.9% (n 5 10) and 9.7% (n 5 14) at
concentrations of 322 and 347 mg/ml, respectively.
Hydroxyproline. Total hydroxyproline (OH-Pro) in blood
reflects collagen breakdown and thus catabolic mechanisms. It was measured using an HPLC method published
before (Claus et al., 2007). Coefficients of variation were
2.7% (n 5 10) for intraassay and 9.1% (n 5 14) for interassay variations in samples containing 22.6 and 20.9 mg/ml,
respectively.
Protein synthesis by infusion of 13C-leucine. Determination
of protein synthesis by infusion of 13C-leucine is an established method (Waterlow et al., 1978). It is based on a
constant infusion of leucine up to a constant equilibrium so
that the maintenance of a plateau during ongoing infusion
represents leucine used for protein synthesis.
To obtain background values for 13C/12C-enrichment, a
sample of heparinized blood was taken before giving the
bolus of leucine described below. A bolus of 30 mg L-[1-13C]
leucine (Euroisotope, Saarbrücken, Germany) dissolved in
5 ml saline was administered intravenously, followed by
continuous infusion at the rate of 30 mg/h for 6 h (150 ml/h)
via the implanted catheter using a peristaltic pump. Blood
samples were taken at 30 min intervals during the 6 h
infusion period. To measure the exact concentration of
leucine solution infused to each pig, an aliquot of 10 ml was
taken from the residual solution at the end of the experiment, and all animals were weighed before slaughter.
Leucine was isolated from plasma and derivatization was
performed using the test kit EZ:faast (Phenomenex,
Aschaffenburg, Germany). In brief, plasma samples were
acidified by 1 M HCl (40 ml/100 ml plasma), amino acids
were extracted by a cation exchanger, eluted and derivatized using propyl chloroformate. For quantification, norvaline was added as internal standard. GC-MS (GC-17A
coupled to a QP 5050, Shimadzu, Duisburg, Germany) was
conducted in the single ion mode (m/z 158 for internal
1217
Total body fat content. Because GH primarily changes the
lean/fat ratio, anabolic parameters are insufficient and the
additional determination of body fat with the D2O method
was included. Application of D2O, measurement of isotopic
enrichment by infrared spectroscopy and calculations
including coefficients of variations were published recently
(Claus et al., 2007).
Statistics
Data were analysed using Statistical Package for the Social
Sciences (SPSS v.13.0; Chicago, IL, USA). Data in figures are
presented as means 6 s.e. of six animals per group, with
day 0 being the day of the first immunization and day 28
the day of the second immunization. In the tables, data for
IGF-1, urea, OH-Pro and fat content are presented for two
phases. Phase 1 comprised values from day 0 to day 27;
phase 2 comprised values from day 39 to day 72 as similarly
reported earlier (Claus et al., 2007). Days 28 to 38 were not
included because of transient increasing or decreasing
values due to the onset of immunization effects. Trend
analysis was performed as described by Neumann (1941)
and Hart (1942). Changes in titre were tested as described
previously (Claus et al., 2007). A linear mixed model with
repeated measures was used to compare barrows and
immunized barrows. Fixed effects (group and phase) and
their interaction were tested for statistical significance.
Replicate was included as a random effect. For none of the
parameters investigated, a significant replicate effect was
found (estimate of covariance parameter by the Wald test).
Interactions were significant for FSH and antibody titres.
Results
Changes in titre of antibodies against GnRH are shown in
Figure 1a and b. In immunized barrows (Figure 1a), antibody titres rose rapidly and peaked seven days after
administration of the second dose of Improvac. Titres then
declined until the end of the study on day 72 after initial
vaccination (P , 0.05; trend analysis). Nevertheless, on day
72, titres were still higher than they had been before the
second immunization (P , 0.001). Differences between
both groups and phases were highly significant. Control
1218
5
(a)
4
E (450 nm)
standard, m/z 200 and 201 for leucine) under conditions
described by the manufacturer of the test kit. For quantification of 13C-leucine in infusion solutions, an external
standard containing 200 nmol/ml was processed as described above and the leucine content calculated using the
internal standard, after which the infusion rate was estimated. To determine 13C-enrichment, peak areas for m/z
201 were related to m/z 200 and plotted against time of
sampling. Intraassay coefficient of variation was 2.75%.
Constant enrichment was obtained from 3 h to 6 h after
onset of infusion and was used to calculate the total leucine
flux together with the infusion rate (see above). For calculation, a percentage of 7.6% leucine in total protein was
assumed (Waterlow et al., 1978; Eggum, 1989).
3
2
1
0
0
12
24
36
48
60
72
12
24
36
days
48
60
72
(b)
1
0
0
Figure 1 Antibody titre in immunized barrows (a) and control barrows (b).
First immunization was on day 0 (18 weeks of age). Second immunization
was on day 28 (22 weeks of age). Closed circles represent animals from
replicate 2, open circles animals from replicate 1.
barrows (Figure 1b) had low extinctions around 0.3
throughout the experimental period, comparable to values
in immunized animals before they were given a second
dose of Improvac.
A decrease in LH to values near the detection limit
within 5 days was observed after the second immunization
(Figure 2a; P , 0.01). The decrease in LH between the first
and second immunization was significant in four of the six
pigs as shown by trend analysis (P , 0.05). Plasma LH in
control barrows (Figure 2b) remained at about 0.5 ng/ml
throughout the experiment.
FSH determination (Figure 3a) revealed decreasing values
already before the second immunization in five of the six
immunized barrows (P , 0.05; trend analysis). After the
second dose of the vaccine, FSH decreased from 1.3 to
0.5 ng/ml (P , 0.001) and remained at this level until the end
of the study. Control barrows (Figure 3b) began with levels
of about 2 ng/ml and these did not decrease significantly.
Testosterone values in all animals remained below the
detection limit of 0.05 ng/ml throughout the experiment
(data not shown). There was no significant difference
between vaccinated and unvaccinated barrows.
The GH parameters are given in Table 2. Mean GH concentrations decreased significantly (P 5 0.01) from the first
window at age 19 weeks (1 week after first vaccination)
1.0
3.5
(a)
(a)
3
0.8
FSH [ng/mL]
LH [ng/mL]
2.5
0.6
0.4
2
1.5
1
0.2
0.5
0
0.0
0
12
24
36
48
60
0
72
12
24
days
1.0
36
48
60
72
48
60
72
days
3.5
(b)
(b)
3
0.8
FSH [ng/mL]
LH [ng/mL]
2.5
0.6
0.4
2
1.5
1
0.2
0.5
0
0.0
0
12
24
36
48
60
72
0
12
24
36
days
days
Figure 2 LH concentrations (mean 6 s.e.) in immunized barrows (a) and
control barrows (b). First immunization was on day 0 (18 weeks of age).
Second immunization was on day 28 (22 weeks of age).
Figure 3 FSH concentrations (mean 6 s.e.) in immunized barrows (a) and
control barrows (b). First immunization was on day 0 (18 weeks of age).
Second immunization was on day 28 (22 weeks of age).
to the second window at age 24 weeks (2 weeks after the
second vaccination) both in control barrows and in immunized barrows. The decrease was 18% in the control barrows and 22% in the immunized barrows. While maxima
decreased in the same way (P , 0.015) the base levels
remained unchanged. The pulse frequency was lower in the
second window compared with the first one (P 5 0.048).
Nevertheless, there were no significant differences between
the two treatment groups in any of the GH parameters
evaluated, in either the first or second sampling windows.
IGF-1 (Table 3) tended to decrease due to immunization
but was not significantly different from the controls
(P 5 0.067).
Data for metabolic parameters are also presented in
Table 3. Compared to phase 1, urea concentrations in
phase 2 were increased by 15 mg/ml in both groups (not
significant) while differences between groups exist
(P 5 0.002). There was a significant decrease in OH-Pro by
about 10% after the second immunization (P 5 0.001).
Protein synthesis values were 5.16 and 5.55 g/kg BW per
day for barrows and immunized barrows, respectively, and
were not significantly different. Comparison of deuterium
oxide applications in weeks 20 and 25 demonstrated that
fat content tended to increase by 15% in this period
(P 5 0.073) and there was no statistical difference between
the groups.
Discussion
In an earlier study (Metz and Claus, 2003), using immunocastrated entire male pigs at ages of 10, 16 and 23 weeks,
we found that GH concentrations in blood plasma at
23 weeks were identical to entire control boars (both
4.09 ng/ml). In contrast, GH concentrations in blood plasma
of surgical castrates only reached 2.88 ng/ml, and thus were
42% lower than the other two groups. The maintenance of
1219
Table 2 Characterization of GH concentrations in blood plasma of barrows and immunized barrows in 24-h windows at an age of 19 and 24 weeks
Group
Barrows
Immunized barrows
Window week
19
24
19
24
Group
Window
Mean level (ng/ml)
2.53 6 0.12
2.06 6 0.07
2.21 6 0.17
1.72 6 0.12
n.s.
0.01
Basal level (ng/ml)
Maximum (ng/ml)
1.66 6 0.09
1.43 6 0.19
1.43 6 0.16
1.16 6 0.10
n.s.
n.s.
5.36 6 0.48
4.27 6 0.71
4.95 6 0.26
3.95 6 0.25
n.s.
0.015
Pulses (n/24 h)
4.50 6 0.31
3.50 6 0.39
4.33 6 0.19
3.17 6 0.50
n.s.
0.048
Values are given as mean 6 s.e.
Table 3 Metabolic parameters
Group
Barrows
Immunized barrows
Phase 1
Phase 2
Phase 1
Phase 2
Group
Phase
IGF-1 (ng/ml)
Urea (mg/ml)
Hydroxyproline
(mg/ml)
Protein synthesis
(g/kg per day)
Fat content (%)
145.1 6 17.6
122.1 6 14.1
139.5 6 9.0
133.8 6 10.0
n.s.
0.067
328 6 8
343 6 6
378 6 9
393 6 5
0.002
n.s.
23.2 6 0.3
20.4 6 0.5
23.6 6 0.4
21.7 6 0.4
n.s.
0.001
–
5.16 6 0.31
–
5.55 6 0.81
n.s.
–
17.7 6 2.9
22.1 6 1.1
19.0 6 1.7
21.2 6 0.7
n.s.
0.073
Phase 1 refers to day 0 (day of first vaccination at age 18 weeks) to day 27 while phase 2 comprise day 39 to day 72. Protein synthesis was determined on
the last day of phase 2, while fat content was measured on day 2 and day 49.
Values are given as mean 6 s.e.
GH concentration after immunocastration was recently confirmed in another study (paper in preparation; boars: 3.57 ng/ml;
immunized boars: 3.24 ng/ml; not significant). These results
indicated a systematic difference in GH concentrations
between surgically castrated and immunocastrated pigs. It
is well known that GnRH and thus gonadotropins are elevated in barrows due to the absence of negative feedback
by steroids. Since surgically castrated and immunocastrates
have low steroid values, we hypothesized that there may be
an interaction between elevated GnRH and GRH, which
might be abolished by GnRH vaccination of barrows.
In contrast to our earlier study with boars, gonadotropin
concentrations in barrows already decreased in response to
the first vaccination, which is known to lead to low levels of
immunoglobulins primarily represented by IgM. Such an
effect was also found previously (Oonk et al., 1998; Turkstra
et al., 2001 and 2002). It is likely that our assay system did
not detect low levels of IgG and did not detect IgM at all
because it was raised against IgG.
The difference in gonadotropin reaction between boars
and barrows might be due to the absence of negative
steroid feedback in the barrows, which thus have higher
gonadotropin concentrations in the beginning and are
probably more sensitive to low levels of immunoglobulins.
Detailed mechanisms of a possible interaction of the
immune system and hormonal feedback regulation have not
been investigated. Such an assumption, however, is supported by the maintenance of an intermediate LH level of
about 0.2 ng/ml until the second vaccination, which corresponds to the physiological LH concentration in boars
1220
(Wagner and Claus, 2004; Claus et al., 2007). The second
vaccination then led to a rise of antibody titre and to a
further decrease of LH down to base levels. Interestingly,
FSH also decreased continuously after a latency period of
about 10 days after the first immunization. In earlier studies
we found no change in FSH concentrations following
immunization of boars: levels remained the same (0.5 ng/
ml) in immunized and control boars (Wagner and Claus,
2004). In the present study, barrows of both groups had
higher FSH concentrations due to the absence of negative
steroid feedback. Immunization clearly decreased FSH but
values were again stabilized at 0.5 ng/ml. With regard to
FSH regulation, differences exist with other species. Both
the function of activin from the anterior pituitary and the
existence of a hypothalamic releasing hormone, other than
GnRH, were assumed (Kauffold et al., 2005). This does not
exclude the fact that GnRH plays a role in stimulating the
high FSH concentrations due to the absence of negative
feedback in barrows. From other species it is known that
GnRH and activin interact (Liu et al., 1996; Shupnik and
Weck, 1998; Gregory et al., 2005) so that high GnRH
concentrations might be paralleled by elevated activin.
Because activin in turn inhibits the synthesis and release of
GH (Childs and Unabia, 1997), these effects might also
explain lower GH levels in surgical castrates. The comparison of the two windows in the immunized barrows, one
during high and one during low FSH, however, did not lead
to different GH concentrations so that lower levels of GH in
surgical castrates do not support the assumption of activin
effects even if this substance was not measured in this
study. Furthermore, GH secretion was not influenced by
immunization of barrows, so the hypothesis of an interaction
between the releasing hormones GnRH and GRH in the
hypothalamus or pituitary also could not be substantiated.
As many of the metabolic effects of GH are mediated by
IGF-I, levels of this hormone were also determined and
again revealed no differences between barrows and
immunized barrows. As expected, metabolic parameters
confirm the absence of an effect of immunization on GH in
barrows. In both groups, however, the age-dependent
influence is obvious such as the increase of urea concentrations and the decrease of OH-Pro levels in blood
plasma as it is known from other studies (Miller et al.,
1990). The fat content also increased with age, as expected.
It is concluded that interactions between GnRH and GRH in
the hypothalamus or the pituitary do not explain why GH
concentrations in surgically castrated males are lower than
in GnRH immunized boars. It might be that boars, which
remain intact until GnRH immunization, e.g. at 18 weeks,
react differently compared to those that were castrated
during the first week of life. Indeed, it is known from
rodents that a later ‘male pattern’ of GH secretion is
imprinted by gonadal steroids early postnatally (Jansson
et al., 1985). Such a transient increase of gonadal steroids
in pigs is known to occur about 3 to 4 weeks after birth
(Schwarzenberger et al., 1993).
Acknowledgements
We would like to thank Bill Dunne, Mohammed Mecellem and
Claudia Fischinger for animal care; Stefanie Mayer for her
analytical talent, Anna Wagner for supervising the LH and FSH
determinations, and Herbert Steingass and Mrs Haller for the
help during D2O analysis. The study was financially supported
by Pfizer, Sandwich, UK. We also thank Michael Pearce for
critical suggestions and discussions.
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1222
2.3
Effects of two levels of feed allocation on IGF-I concentrations and metabolic
parameters in GnRH-immunized boars
(A Bauer, M Lacorn, R Claus
Journal of Animal Physiology and Animal Nutrition 2009, 93: 744-753)
Article included in consent of Wiley-Blackwell.
48
DOI: 10.1111/j.1439-0396.2008.00860.x
ORIGINAL ARTICLE
Effects of two levels of feed allocation on IGF-I concentrations
and metabolic parameters in GnRH-immunized boars
A. Bauer, M. Lacorn and R. Claus
FG Tierhaltung und Leistungsphysiologie, Institut für Tierhaltung und Tierzüchtung, Universität Hohenheim, Stuttgart, Germany
Keywords
IGF-I, immunocastration, boar, energy supply,
growth
Correspondence
Prof. Dr. R. Claus, FG Tierhaltung und
Leistungsphysiologie, Institut für Tierhaltung
und Tierzüchtung, Universität Hohenheim,
Garbenstraße 17, Stuttgart, 70599,
Germany. Tel: 0049 711 459 22455;
Fax: 0049 711 459 22498;
E-mail: [email protected]
Received: 30 January 2008;
accepted: 22 July 2008
First published online: 18 November 2008
Summary
Immunocastration of boars leads to a maintenance of growth harmone
(GH) and a loss of anabolic hormones [androgens, oestrogens, insulinlike growth factor (IGF-I)] but an increase of voluntary feed intake. The
aim of the experiment was to clarify whether IGF-I is increased by
increasing feed supply in immunocastrated boars leading to improved
anabolism. Two groups of six boars were given 2 or 3 kg of feed
(13.5 MJ ME/kg) daily from 18–28 weeks of age. Because in boars feed
intake is limited by gonadal hormones, a group with further increased
feed supply could not be included. Until week 22 (second vaccination)
gonadal steroids in blood were normal but dropped rapidly thereafter.
Growth harmone levels did not change following vaccination. Pigs allocated 3 kg feed had 28% higher circulating IGF-I after the second
immunization compared with pigs fed 2 kg feed daily. Higher IGF-I was
associated with increased weight gain (682.4 g/day vs. 466.7 g/day;
p < 0.01) and protein synthesis (13C-leucine infusion; 405 g/day vs.
247 g/day, p < 0.01). Protein breakdown (urea) was not different. Body
fat (D2O) decreased in the low feed group from 15.2% (week 19) to
6.1% (week 25). In the high feed group it remained at the level found
before second vaccination (13.7% vs. 15.0%). It is concluded that in the
phase of reduced testicular steroids which inhibit appetite it is possible
to increase feed intake which in turn increases IGF-I and protein deposition without accumulating excessive fat.
Introduction
Boars are well known to have a higher anabolic
potential compared with barrows (castrated males;
van Lunen and Cole, 1996; Campbell et al., 1985)
which leads to improved fattening performance and
carcass characteristics. Surgical castration, however,
is still performed to avoid the unpleasant boar taint
which is not acceptable to many consumers.
Currently, immunological castration is discussed as
an alternative to surgical castration. It is based on
immunization against gonadotropin releasing harmone (GnRH) so that luteinizing harmone (LH)
release from the pituitary is blocked, and in
744
consequence, testicular Leydig cells do not synthesize sex steroids (Claus et al., 2007). Improvac
(Pfizer Animal Health, Karlsruhe, Germany) is an
antigen which is available in several countries.
Immunization is performed by giving two injections
of the antigen at least 4 weeks apart. Seven days
after the second vaccination, metabolism is adapted
to the lack of anabolic gonadal steroids. In the
period between second vaccination (approximately
22 weeks of age) and slaughter (at approximately
28 weeks of age), performance data and anabolic
metabolism do not differ from conventional barrows
although GH is higher in immunized pigs, but this
time must be given to allow decrease of boar taint
Journal of Animal Physiology and Animal Nutrition 93 (2009) 744–753 ª 2008 The Authors. Journal compilation ª 2008 Blackwell Verlag GmbH
Feed allocation and IGF-I in GnRH-immunized boars
from body fat (Claus, 1976). Anabolic steroids which
are absent during this period, include androgens,
e.g. testosterone and nortestosterone as well as high
amounts of testicular oestrogens, which in the boar
by far exceed levels measurable in oestrous sows
(Claus and Hoffmann, 1980). Both androgens and
oestrogens contribute to an improved anabolic performance by increasing nitrogen retention to a level
approximately 20% higher than in barrows (Metz et
al., 2002). Mode of actions of androgens and oestrogens, however, differ.
Androgens lead to muscle fibre hypertrophy by
stimulating muscle protein synthesis and improving
re-utilization of amino acids. In addition, muscle
protein degradation and cell apoptosis are decreased
after androgen application to castrated males. This
effect is mainly explained by formation of heterodimers between androgen and glucocorticoid receptors, which decrease the catabolic action of cortisol
(Bhasin et al., 2003; Chen et al., 1997). Such glucocorticoid receptors also exist in pig muscles (Claus
et al., 1996).
The anabolic effect of oestrogens is primarily
explained by stimulating concentrations of the insulin-like growth factor-I (IGF-I) in peripheral blood.
This mitogenic factor is expressed in almost every
tissue, including muscle tissue (Novakofski and
McCusker, 1993; Reeds et al., 1993; Kamanga-Sollo
et al., 2004), but is also secreted as a hormone from
the liver in response to GH stimulation (Breier et al.,
1989; Etherton, 1989; Novakofski and McCusker,
1993). In the liver, oestrogens upregulate hepatic
GH receptors and thus boost GH-dependent IGF-I
expression (Claus et al., 1992a). Insulin-like growth
factor-I then is the most potent stimulus for protein
synthesis.
Active immunization shifts metabolism of boars to
a less pronounced anabolic state following the
decrease of androgens and oestrogens (Claus et al.,
2007). It is additionally known that sex steroids
decrease voluntary feed intake as shown by comparisons of boars and barrows and experimentally either
by testosterone or estradiol administration to barrows (Claus and Weiler, 1987; 1994).
Insulin-like growth factor-I formation is very sensitive to feed intake (Morovat et al., 1994; Owens
et al., 1999) mainly because of the stimulating function of carbohydrates with a high glycaemic index
such as glucose or starch (Reeds et al., 1993). In
consequence, it is possible that the decrease of testicular hormones because of immunization leads to an
increased voluntary feed intake which in turn
elevates IGF-I. Such a mechanism would allow
development of feeding strategies which increase the
anabolic potential again in finishing immunocastrates. Therefore it was the aim of this study to clarify whether the level of feed supply in the finishing
period of immunocastrates influences IGF-I and
improves parameters which characterize metabolic
reactions.
Materials and methods
Study design
German Landrace boars (n = 12) were obtained from
the swine herd of the University of Hohenheim.
They were randomly divided into two experimental
groups (each n = 6) and kept in individual crates
measuring 3 m · 2.5 m. At 18 weeks of age they
received the first 2 ml dose of the GnRH-antigen
(Improvac). A single dose of Improvac has no
effect on testicular function and metabolic parameters (Claus et al., 2007). In the following 4 weeks
(phase 1; between 18 and 22 weeks of age), hormones (GH, LH, IGF-I, testosterone and estradiol)
and metabolic parameters were measured in blood
plasma to characterize the boar-specific endocrine
and metabolic status. At 22 weeks of age in accordance with the manufacturer‘s recommendations,
the second dose of Improvac was given to immunocastrated pigs and the responses of hormonal and
metabolic parameters were determined.
Animals were fed from an age of 18 weeks till
slaughter at 28 weeks with a ration containing
13.5 MJ ME/kg (crude protein 16.7%; Table 1). The
main energy source was starch (443 g/kg) with a
high glycaemic index which is the prerequisite to
provoke the expected effect on IGF-I. Group 1
received 2 kg per day, corresponding to 27 MJ per
day, which is below recommendations (32 MJ per
day) at a body weight of 80 kg. Group 2 received
3 kg feed daily so that total supply (40.5 MJ
per day) was above requirement (Ausschuss
Ernährungsphysiologie, 2006). A third group with
Table 1 Feed composition
Component
Wet weight (g/kg)
Barley
Triticale
Wheat
Pea
Soybean meal
Calcium carbonate
Soybean oil
373
200
170
70
150
25
2
10
Journal of Animal Physiology and Animal Nutrition. ª 2008 The Authors. Journal compilation ª 2008 Blackwell Verlag GmbH
745
further increased feed supply was not realistic,
because appetite is limited because of testicular steroids. The pigs were fed twice daily at 8:00 and
15:00 hours with 1 kg (group 1) and 1.5 kg (group
2) respectively. During the experimental period, feed
intake was monitored twice daily by weighting residual feed. Weight gain was determined every week.
The whole experiment had been approved by the
Animal Welfare Committee of the state of Baden–
Württemberg.
Cannulation and blood sampling
Each pig was fitted with a permanent cephalic vein
catheter under general anaesthesia at an age of
17 weeks as described earlier (Claus et al., 1990). At
that age, body weight varied between 51 and 60 kg
in group 1 and 49 and 62 kg in group 2, so that
mean body weight did not differ significantly
between group 1 and 2. The animals had completely
recovered from cannulation 2 days after surgery.
Blood samples were drawn daily at 9.00 hours in
heparinized vials and the plasma stored deep-frozen
at )20 C until measuring the different parameters.
After each sampling, the catheters were rinsed using
sterile saline with 0.1% heparin.
Protein synthesis
Protein synthesis was determined by the 13C-leucine
infusion method. The principles were described earlier (Waterlow et al., 1978). Details are given in a
preceding paper (Bauer et al., 2007). In brief, a constant infusion of 13C-leucine was applied for 6 h
after a single bolus of 13C-leucine which had been
given via the catheter. Blood samples were taken
every 30 min, leucine was isolated, derivatized using
the test kit EZ:faast (Phenomenex, Aschaffenburg,
Germany) and analysed for 13C/12C enrichment by
gas chromatography–mass spectrometry (GC–MS).
Constant enrichment was obtained between 3 and
6 h of infusion and was used to calculate the total
leucine flux and subsequently protein synthesis
using the method of Waterlow et al. (1978). Intraassay coefficient of variation was 2.75%.
Body fat determination
To mirror changes in body fat content, the deuterium oxide (D2O) method was used (Tissier et al.,
1978; Susenbeth, 1984). The D2O windows were
performed at an age of 20 and 25 weeks. Application, measurement of isotopic enrichment by
746
infrared spectroscopy and calculation were already
described (Claus et al., 2007). Intra- and interassay
coefficients of variation were 0.2% (n = 5) and 2.2%
(n = 10) respectively.
Analytical methods
GnRH-antibody titre
Relative concentrations of anti-GnRH antibodies
were determined using a microtitre plate assay as
previously described (Bauer et al., 2007). In brief,
plates were coated with GnRH (Bachem, Weil am
Rhein, Germany), incubated with diluted blood
plasma (1:1600) and subsequently incubated with a
second antibody-enzyme-conjugate (P 0164, specific
for all subclasses of swine IgG; Dako, Hamburg,
Germany). Horse radish peroxidase was used as the
enzyme and tetramethylbenzidine/H2O2 as substrate.
Extinction was measured at 450 nm. Interassay coefficients of variation were 2.56% for an extinction
of 3.67 and 5.44% for an extinction of 2.33 (n = 5).
Luteinizing hormone and growth hormone
The experimental period lasted 10 weeks, so high
frequent sampling of blood for measuring the
pulsatile LH and GH secretion patterns was not possible. Instead, they were measured by radioimmunoassay (RIA) in samples taken every third (LH) or
second (GH) day (Wagner and Claus, 2004; Claus
et al., 1990) which is sufficient to reflect major
changes over this prolonged period.
The specific LH antiserum (AFP 15103194) was
provided by Dr. Parlow (NIDDK, Torrance, CA, USA)
and a highly purified pLH standard (AFP 11043B)
from the same source was used for radio-iodination
and for calibration. Interassay coefficients of variation were 7.3% and 9.1% for concentrations of 380
and 40 pg/ml respectively. The lower limit of detection was 24 pg/ml and average recovery was 92%
for concentrations of 250 pg/ml.
A specific GH antiserum (AFP422801) was also
provided by Dr. Parlow (NIDDK, Torrance, CA,
USA) and a highly purified pGH standard
(AFP10864P) was used for radio-iodination and
calibration. Repeatability was determined by
measuring spiked samples on consecutive days and
the interassay coefficient of variation was found to
be 7.5% for levels of 3.4 ng/ml (n = 6). The
intraassay coefficient of variation was 3.6% for
concentrations of 4.6 ng/ml (n = 10) and the
recovery was above 96% for spiked samples
(1.5–6 ng/ml).
Testosterone
Radioimmunological determination of testosterone
in plasma samples of every third day after solvent
extraction was performed as published earlier (Bauer
et al., 2007). Coefficient of variation between days
was 8% at a concentration of 1 ng/ml (n = 6). The
lower limit of detection was 0.05 ng/ml. The extraction yield was 90%.
17b-Estradiol
Concentrations of 17b-estradiol in blood plasma
were determined every third day as described before
(Claus et al., 1983). The antiserum was highly specific for 17b-estradiol and showed no cross-reactivities with other oestrogens. Interassay coefficient of
variation was 9.4% for concentrations of 84 pg/ml
(n = 9). Recoveries were above 95%.
Androstenone
Levels of androstenone (5a-androst-16-en-3-one) in
blood samples which had been drawn every third
day were determined after n-hexane extraction by
GC–MS analysis in the single-ion mode as described
previously (Claus et al., 2007). Repeatability was calculated by measurement of a pool sample either on
the same day (n = 4; intraassay coefficient of variation was 3.7% at a concentration of 1.0 ng/ml) or
on consecutive days (n = 13; interassay coefficient of
variation was 8.2% for 1.15 ng/ml).
Insulin-like growth factor-I
To determine IGF-I in plasma samples from every
second day, a double-antibody RIA was used which
includes an HCl/ethanol extraction to separate IGF-I
from its binding protein. The specific antiserum was
raised in rabbits (Claus et al., 1992a,b). The IGF-I
standard for radio-iodination and for calibration was
obtained from Gro Pep (CU020; Adelaide, Australia).
The average recovery for spiked samples was approximately 70% for concentrations between 50 and
400 ng/ml. The coefficients of variation within and
between days were 7% (n = 10; 186 ng/ml) and 5%
(n = 7; 74 ng/ml) respectively.
Urea
Urea was measured in plasma to characterize protein
degradation as a marker for nitrogen excretion,
because we found earlier that transfer of pigs into
metabolic crates for N-retention studies is sufficient to
shift metabolism to a more catabolic status. The
method was described in detail earlier (Claus et al.,
2007). In brief, plasma was pre-incubated with
glutamic dehydrogenase to remove ammonia by
reaction with oxoglutarate, adenosine diphosphate
and reduced forms of nicotinamide adenine dinucleotide. After addition of urease released ammonium was
quantified at 340 nm. Quantification in blood samples
of every second day was performed and interassay
coefficient of variation was 3.6% (n = 14) in a sample
containing 341 lg/ml.
Statistics
Data were analysed using the Statistical Package
for the Social Sciences (spss version 13.0; Chicago,
IL, USA). Data in figures and tables are presented
as means SEM of six animals per group, with
day 0 being the day of the first immunization and
day 28 the day of the second immunization. For
statistical analysis two phases were calculated as
reported earlier (Claus et al., 2007): phase 1 comprised values from day 0 to day 27; phase 2 comprised values from day 39 to day 72. Days 28–38
were excluded because they represented the period
of transient increasing or decreasing values because
of the onset of immunization effects. Trend analysis was performed as described by Neumann (1941)
and Hart (1942). Changes in titre development
were tested as described previously (Claus et al.,
2007). Differences between phases within one
group were tested using paired Student’s t-test,
whereas differences between groups were analysed
with unpaired Student’s t-test. Possible effects that
influence results, such as time of birth, weight at
18 weeks of age and genetics were excluded by
the study design as described above.
Results
Confirmation of immunization success
The development of the relative concentration of
GnRH antibodies in immunized animals is
summarized in Fig. 1 for both groups and compared
with the course of GH concentrations. Concentration
of GnRH antibodies rose rapidly 4 days after second
immunization (day 28). Maximal levels were
reached on day 37. Thereafter relative concentration
of GnRH antibodies decreased significantly (trend
analysis; p < 0.05), but mean extinctions of the
animals during the last week of the experiment were
still elevated compared with mean values of phase 1
747
8
3
7
GH (ng/mL)
E (450 nm)
2.5
2
1.5
1
6
5
4
3
2
0.5
0
1
0
6 12 18 24 30 36 42 48 54 60 66 72
Days
0
0
6 12 18 24 30 36 42 48 54 60 66 72
Days
Fig. 1 Development of relative concentration of gonadotropin releasing harmone antibodies (left: mean extinctions SEM for both groups;
n = 12) and mean growth harmone (right) both for immunized boars of group 1 (black squares: fed with 2 kg) and group 2 (open squares: fed with
3 kg). First immunization was on day 0 (18 weeks of age). Second immunization was on day 28 (22 weeks of age).
(p < 0.01). Immunization had no significant effect
on the course of GH concentrations.
Consequent to the increase in relative levels of
antibodies against GnRH, concentrations of LH and
androstenone were low until slaughter and are
summarized in Table 2 to further confirm the
effectiveness of immunization. LH dropped significantly after second immunization (phase 2) within
8 days from mean concentrations of 100 pg/ml
before second immunization (phase 1) to values
near the detection limit of 24 pg/ml in both group
1 and 2 (p < 0.05 for both groups). Similarly,
levels of androstenone reached minimum values
8 days after second immunization in group 1
and 2.
residues of approximately 100–500 g/day occasionally remained under the influence of testicular
hormones before second vaccination. With the onset
of the immunization effect during phase 2 and thus
the drop of gonadal steroids, appetite increased and
feed was completely consumed.
Irrespective of the group, all animals entered the
experiment with the same body weight and
increased weight in a linear mode during the whole
experimental period (coefficients of determination in
a range of r2 = 0.94–0.99). Daily weight gain was
579 and 867 g/day for groups 1 and 2, respectively,
over the whole experimental period (age 18–
28 weeks). Weight gain started to differ between the
two groups 1 week after second vaccination.
Performance data
Metabolically active hormones and parameters of
metabolism
Monitoring of feed intake revealed that in the 2 kg
group, no feed residues were left by the pigs over
the whole experimental period. In the 3 kg group,
Table 2 LH and androstenone concentrations of immunized boars
fed 2 kg/day (group 1) and immunized boars fed 3 kg/day (group 2) in
phase 1 (before second immunization) and phase 2 (after second
immunization)
Parameter
Phase
Group 1
Group 2
LH (pg/ml)
1
2
1
2
80 10*
30 0
1.54 0.30*
0.18 0.03
120 20*
40 10
0.93 0.19*
0.16 0.03
Androstenone (ng/ml)
LH, luteinizing harmone.
Asterisks refer to differences between phases within one group
(*p < 0.05). Comparison between groups within the same phase
revealed no differences.
Mean SEM are given.
748
Other parameters are given as continuous profiles
separately for the two feeding groups in Figs 2–5.
A comparison of differences between groups after
second immunization in phase 2 is summarized in
Table 3. Measurement of testosterone (Fig. 2) in
animals from groups 1 and 2 showed decreasing
values
8 days
after
second
immunization
(p < 0.01). Minimal values corresponded to the
limit of detection of the assay and thus, of course,
were not significantly different between the two
groups in phase 2. Mean values in phase 1 also
did not differ significantly.
Concentrations of 17b-estradiol (Fig. 3) did not
differ significantly between groups in phase 1 (group
1: 71 pg/ml; group 2: 46 pg/ml). After second vaccination, 17b-estradiol dropped in both groups (Fig. 3)
and mean values thereafter were not significantly
different between groups (Table 3).
2.5
Testosterone (ng/mL)
Testosterone (ng/mL)
2.5
2.0
1.5
1.0
0.5
0.0
0
2.0
1.5
1.0
0.5
0.0
6 12 18 24 30 36 42 48 54 60 66 72
0
6 12 18 24 30 36 42 48 54 60 66 72
Days
Days
Fig. 2 Testosterone concentrations (mean SEM; n = 6) for group 1 (left; immunized boars fed with 2 kg) and group 2 (right; immunized boars
fed with 3 kg). First immunization was on day 0 (18 weeks of age). Second immunization was on day 28 (22 weeks of age).
110
110
100
100
90
90
80
80
70
70
60
60
50
50
40
40
30
30
0 6 12 18 24 30 36 42 48 54 60 66 72
0 6 12 18 24 30 36 42 48 54 60 66 72
Days
Days
225
225
200
200
IGF-1 (ng/mL)
IGF-1 (ng/mL)
Fig. 3 17b-estradiol concentrations (mean SEM; n = 6) for group 1 (left; immunized boars fed with 2 kg), group 2 (right; immunized boars fed
with 3 kg). First immunization was on day 0 (18 weeks of age). Second immunization was on day 28 (22 weeks of age).
175
150
125
100
75
175
150
125
100
75
0 6 12 18 24 30 36 42 48 54 60 66 72
0 6 12 18 24 30 36 42 48 54 60 66 72
Days
Days
Fig. 4 Insulin-like growth factor concentrations (mean SEM; n = 6) for group 1 (left; immunized boars fed with 2 kg), group 2 (right; immunized
boars fed with 3 kg). First immunization was on day 0 (18 weeks of age). Second immunization was on day 28 (22 weeks of age).
Data for IGF-I are shown in Fig. 4 and revealed
levels of approximately 160 ng/ml in phase 1, before
second immunization, for both groups and were not
significantly different. After second immunization,
boars showed decreasing values over a period of
14 days and stabilized at lower concentrations of 86
and 119 ng/ml for group 1 and 2, respectively, corresponding to a difference of 28%. These differences
between the phases were significant (p < 0.05) as
were differences between groups in phase 2 (Table 3).
Courses of urea concentrations are given in Fig. 5.
Until second immunization, both groups remained at
approximately 160 lg/ml, while second immunization led to increasing values within 10 days to mean
concentrations of 253 lg/ml in group 1 and up to
288 lg/ml in group 2 (comparison of phases for both
groups: p < 0.01). In phase 2 differences between
group 1 and 2 (Table 3) were not significant.
Protein synthesis was determined at the end of the
experiment only and is given both in g/day and
749
375
375
325
325
urea (mg/mL)
urea (mg/mL)
275
225
175
275
225
175
125
125
0 6 12 18 24 30 36 42 48 54 60 66 72
Days
0
6 12 18 24 30 36 42 48 54 60 66 72
Days
Fig. 5 Urea concentrations (mean SEM; n = 6) for group 1 (left; immunized boars fed with 2 kg) and group 2 (right; immunized boars fed with
3 kg). First immunization was on day 0 (18 weeks of age). Second immunization was on day 28 (22 weeks of age).
Table 3 Comparison of values in phase 2 for the two groups with different feed intake
Parameter
Group 1 (2 kg)
Group 2 (3 kg)
Significance
Weight gain (g/day)
Testosterone (ng/ml)
17b-estradiol (pg/ml)
IGF-I (ng/ml)
Protein synthesis
(g/kg*day)
(g*day)
Urea (lg/ml)
Body fat content (%)
466.7
0.14
33.1
86.3
43.3
0.00
1.3
5.3
682.4
0.17
28.1
118.5
21.1
0.02
2.7
8.6
p < 0.01
n.s.
n.s.
p < 0.01
2.44
247.1
253.0
6.1
0.19
18.5
26.4
2.4
3.27
404.5
287.8
15.0
0.32
41.9
20.3
3.0
p < 0.05
p < 0.01
n.s.
p < 0.05
IGF-I, insulin-like growth factor; ns, not significant.
body weight in Table 3. Significant differences
(p < 0.05) were found between group 1 (2.44 0.19 g/kg*day) and group 2 (3.27 0.32 g/kg*day)
and even more pronounced (p < 0.001) when giving
protein synthesis in g per day.
Body fat content did not differ significantly
between groups in phase 1 (group 1: 15.2%, group
2: 13.7%). but was significantly different in phase 2
(group 1: 6.1%, group 2: 15.0%; Table 3). There was
a significant decrease in body fat from phase 1 to
phase 2 in group 1 (p < 0.05), but no significant differences between phases in group 2.
Discussion
A variety of short-term or long-term effects on
hepatic IGF-I expression and in consequence circulating levels of IGF-I are well described. They
include hormonal effects such as concentrations of
GH (Novakofski and McCusker, 1993) and estradiol
(Claus et al., 1992a) but also nutritional effects
such as the content of carbohydrates or specific
amino acids (Miuray and Noguchi, 1992) in the
750
diet and the amount of feeding. After fasting, concentrations of IGF-I decrease and increase again
during the refeeding period in human adults (Isley
et al., 1983), rats (Phillips et al., 1978), chickens
(Lauterio and Scanes, 1987) and neonatal pigs
(Jones and Campion, 1986). Short-term changes
can be effected by changes of ambient temperature
without changes to feed intake (Morovat et al.,
1994).
In this study, GnRH immunized boars were used
as a model because immunization abolishes testicular
steroids so that IGF-I expression in the liver and
inhibition of appetite are decreased. The effect of
gonadal steroids to lower feed intake was shown earlier by supplementing barrows with either oestrogens or androgens (Claus and Weiler, 1987). This
function probably is mediated via steroid receptors
in the hypothalamus because it was shown e.g. that
the anorectic effect of oestrogens may be mediated
by decreasing neuropeptide-Y release in the hypothalamus (Bonavera et al., 1994). Additionally,
many other factors are currently discussed as potent
regulators of feed intake.
Following immunization, levels of GH are maintained at levels seen in entire boars, whereas surgical
castrates (barrows) have much lower GH concentrations (Metz and Claus, 2003). Hormone determinations in this present study confirm the maintenance
of GH and the abolishment of gonadal steroids, so
that amount of feed was the only variable and its
effect on IGF-I and the metabolic consequences
could be studied separately.
Compared with other studies with either feed
withdrawal or energy supply only for maintenance
(Buonomo and Baile, 1991; Charlton et al., 1993;
Booth et al., 1996; Salfen et al., 2003), our feeding
regime was deduced from energy requirements and
digestibility for finishing rations of pigs at 80 kg live
weight. The average weight of 80 kg coincided with
the second immunization. The moderate feeding differences between groups were more in a physiological range. From earlier studies we know that
voluntary feed intake in 80 kg boars is around 3 kg
(Metz et al., 2002; Bauer et al., 2008).
Data obtained show that both groups of immunized boars after second vaccination reacted indeed
with a decreased IGF-I expression. It was also apparent by comparing IGF-I in phase 1, before onset of
testicular inhibition, and phase 2, when steroid synthesis is inhibited, that the marked decrease in IGF-I
is associated with the decrease in circulating steroids.
During phase 2, even in the 3 kg group, residues of
feed were never left, so that it is reasonable to
assume that feed intake could have been higher.
Such a further increase probably would have
allowed a further growth improvement. At the first
look such an increase would probably lead to
increased fat deposition but we demonstrated using
the deuterium method that fat content in the 3 kg
group did not increase and is still 32% lower compared with barrows of the some weight and feeding
regime (Bauer et al., 2007). At least a part of the
additional energy apparently consumed by group 2
was used for elevated protein synthesis. The IGF-Idependent increase of protein synthesis led to differences of 25% between the two groups as shown by
13
C-leucine infusion, and also by the high-weight
gain. Urea in contrast did not differ between groups.
Urea is an appropriate parameter to monitor protein
degradation (Lopes et al., 2004) caused by glucocorticoids as the main regulator of protein breakdown.
The catabolic effect of glucocorticoids is counteracted
by testosterone, which was absent in both groups of
immunocastrates and thus explains the increase of
urea after second vaccination. Differences in protein
synthesis thus were not paralleled by differences in
breakdown. It was shown earlier that the rate of
protein breakdown generally is independent from
the rate of protein synthesis (Reeds et al., 1980).
Growth harmone alone, without a rise of IGF-I is
not efficient to increase protein synthesis (Novakofski and McCusker, 1993). Because immunocastration
inevitably eliminates oestrogens, an increased supply
of carbohydrates in the form of starch is an alternative agent to couple GH and IGF-I. Similar to estradiol, these carbohydrates are stimulators for GH
receptor expression in the liver (Brameld et al.,
1999). Because GH efficiently inhibits fat synthesis
(Etherton, 2001), surplus energy is allocated to protein synthesis. Under an energy deficit, e.g. as in
group 1, GH is known to exert a powerful lipolytic
effect. In consequence, this mechanism might
explain the remarkable decrease of fat in group 1
but still the maintenance of a notable level of daily
weight gain.
In conclusion it is likely that elevation of feed supply even above 3 kg/day might further improve protein synthesis without excessive fat deposition in
immunocastrates and thus to adapt performance also
after second immunization to boars which are not
castrated.
Acknowledgements
We thank Mohammed Mecellem, Bill Dunne and
Claudia Fischinger for care of the animals and Dr. U.
Weiler who participated in animal surgery. Stefanie
Mayer, Carmen Ostertag and Sybille Knöllinger performed excellent laboratory work, Birgit Deininger
prepared the manuscript. We also thank Herbert
Steingass, Ms Haller, and Ms Brosig for their help
during D2O analysis and Michael C. Pearce for valuable discussion and proofreading of the manuscript.
Financial support was given by Pfizer Animal
Health.
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2.4
Is the early postnatal rise of testosterone responsible for a later male pattern of
growth hormone secretion in pigs?
(M Lacorn, A Bauer, R Claus
Theriogenology 2009, 72: 636-642)
Article included in consent of Elsevier.
60
Available online at www.sciencedirect.com
Theriogenology 72 (2009) 636–642
www.theriojournal.com
Is the early postnatal rise of testosterone responsible for a later male
pattern of growth hormone secretion in pigs?
M. Lacorn, A. Bauer, R. Claus *
Institut für Tierhaltung und Tierzüchtung, Universität Hohenheim, Garbenstrasse 17, 70599 Stuttgart, Germany
Received 8 October 2008; received in revised form 14 March 2009; accepted 27 April 2009
Abstract
Sexual differentiation in Placentalia consists of several consecutive steps during fetal, postnatal, and premature development. It
is known from male rats that an elevation in testosterone synthesis is observable within 2 d of birth, which leads to a male pattern of
growth hormone (GH) secretion with low base levels and high amplitudes compared with that in females. In the male pig, a transient
rise in testosterone concentration occurs about 4 wk after birth, but it is unknown whether it results in a later male pattern of GH
secretion. In this study, male pigs (sus scrofa) were castrated either at 1 wk of age (Group 1, n = 8) or at 6 wk of age (Group 2, n = 8).
Blood was sampled daily via cephalic vein catheters between 17 and 29 wk of age and analyzed for testosterone, GH, insulin-like
growth factor-1 (IGF-1), and urea. High-frequency blood sampling (every 20 min over 24 h) for determination of GH pulsatility was
performed at ages 19 and 24 wk. Total fat content and protein synthesis were determined at age 25 wk and at slaughter, respectively.
Comparing Groups 1 and 2, there were no differences in daily GH concentrations or pulsatile secretion patterns, but in both groups,
mean GH levels and pulsatility decreased from Week 19 to Week 24. Consequently, IGF-1, protein synthesis, urea, and body fat
showed no differences when comparing both groups. It is concluded that the postnatal rise of testicular steroidogenesis in male pigs
is not responsible for the later male pattern of GH secretion.
# 2009 Elsevier Inc. All rights reserved.
Keywords: GH; Pig; Postnatal imprinting; Testosterone
1. Introduction
In Placentalia, differentiation of the bivalent gonadal
structures (anlagen) to either the testes or the ovaries is
determined by the presence or absence of a Ychromosome. Further sex differentiation is then subject
to the principle of basic femaleness. In females, and thus
in the absence of fetal testicular endocrine function, the
primordia of the male tract degenerate and those of the
female system are stabilized [1], whereas in males,
differentiation of internal and external genitalia requires
* Corresponding author. Tel.: +49 711 45922455;
fax: +49 711 45922498.
E-mail address: [email protected] (R. Claus).
0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2009.04.019
androgen synthesis by the fetal testis, which can be
observed as early as 4 wk after fertilization in the pig
[2,3]. In addition to the genital tract, structures of the
central nervous system, specifically the hypothalamus,
are subject to differentiation, leading to later sexdependent differences of brain function. In species with a
long gestation period, these consecutive differentiation
steps are performed before birth. In contrast, species with
a short gestation period such as rats and mice do not
complete differentiation before birth so that it extends to
early postnatal life [4]. Such irreversible mechanisms of
sexual differentiation have to be distinguished from
phenomena without prior sexual differentiation and thus
can also be released by gonadal steroid application in
mature animals [5–7].
M. Lacorn et al. / Theriogenology 72 (2009) 636–642
Anabolic metabolism and pattern of protein and fat
deposition are also subject to sexual dimorphism. The
higher capacity of males to synthesize and deposit protein
is explained both by the anabolic function of androgens
and their effect directly on target tissues [8]. Additionally,
androgens elevate the growth hormone (GH)–insulin-like
growth factor-1 (IGF-1) system and thus stimulate GHdependent growth in males [9,10]. Experiments with rats,
however, suggest that later pulsatile GH secretion is part
of sexual differentiation, imprinted by the presence of
androgens during critical periods before puberty [11]. In
normally developed adult male rats, such a pattern of GH
is characterized by a low baseline but regularly occurring
pulses with high amplitudes [12]. It was shown that the
baseline is not dependent on imprinting mechanisms
because it requires the presence of androgens later in life
and can also be released in females after androgen
substitution [13]. Occurrence and height of GH pulses,
however, were found to depend on differentiation within
48 h of birth. During this period, androgen synthesizing
capacity of the testis is elevated [14]. Castration at 25 d of
age had no effect on pulsatile GH secretion [15].
The assumption of imprinting effects on GH in rats
was further substantiated by Chowen et al. [16] who
found that the number of neurons that secrete the GHreleasing hormone are more abundant in the hypothalamus of adult rats that had been castrated and
androgen-substituted at the day of birth compared with
rats that were castrated and not androgen treated. In
addition to an increased number of neurons, their later
sensitivity to steroids is improved.
In the pig, the course of androgen concentrations in
blood plasma of fetal and postnatal male and female pigs
is well established [2,17,18], and a postnatal transient rise
about 4 wk after birth has been found regularly. This rise
is also assumed to be important for later sexual and social
behavior [19]. It might be that this transient rise of
androgen synthesis has also an effect on the GH secretion
pattern as found in the rodent studies, but this aspect has
not been investigated so far in pigs.
It was the aim of this study to clarify whether the
neonatal rise of testosterone has an effect on
postpubertal male GH secretion pattern in the pig.
2. Materials and Methods
637
surgically castrated at either 1 wk of age (Group 1) or 6
wk of age (Group 2). The castration at age 6 wk was
performed under anesthetic and postsurgical analgesic
treatment.
At age 15 wk, the pigs were brought to the
experimental unit of the University of Hohenheim.
Each pig was kept separately in an individual pen
measuring 3 2.5 m and fed twice daily at 0800 h and
1500 h with a diet containing 16.7% crude protein and
13.5 MJ ME/kg. Details of the diet composition were
given earlier [20]. At 25 wk of age, deuterium oxide was
administered for the later determination of the total
body fat content [21]. They were all slaughtered at 29
wk of age at an average weight of 138 kg, which did not
differ significantly between groups. At the day of
slaughter, the rate of protein synthesis was determined
by infusion of [13C]leucine as described elsewhere [22].
2.2. Blood sampling
For blood sampling (Weeks 17 to 29), each barrow
was fitted with an indwelling cephalic vein catheter at
17 wk of age (range of body weights in Group 1, 54 to
76 kg; in Group 2, 54 to 72 kg) as described earlier [23].
The animals had completely recovered from cannulation 2 d after surgery. Blood samples were drawn daily
at 0900 h in heparinized vials and stored at –20 8C after
centrifugation until measuring the different parameters.
After each sampling, the catheters were rinsed using
sterile saline with 0.1% heparin.
The whole experiment including the late castration
and cannulation had been approved by the Animal
Welfare Committee of the state of Baden-Württemberg.
2.3. Analytical methods
2.3.1. Testosterone
A specific RIA (radio immuno assay) including a
solvent extraction step was used as described earlier [24]
to measure testosterone in plasma samples taken every
third day. Repeatability was determined by measurement
of spiked samples on consecutive days (n = 6), and the
interassay coefficient of variation was found to be lower
than 8% for concentrations between 0.5 ng/mL and 2.5
ng/mL. The lower limit of detection was 0.05 ng/mL. The
average recovery after extraction was above 95%.
2.1. Study design
German Landrace pigs (sus scrofa, n = 16) were
obtained from the swine herd of the University of
Hohenheim. They were randomly divided into two
experimental groups (each n = 8). The animals were
2.3.2. Growth hormone
Growth hormone was determined in samples taken
every second day and, additionally, in window samples
that were drawn every 20 min for 24 h at 19 and 24 wk
of age.
638
and calibration. The intra-assay coefficient of variation
was 9.3% at a concentration of 4.2 ng/mL (n = 10), and
the interassay coefficient of variation was 10% (mean: 3
ng/mL, n = 6). Recovery was above 95% for spiked
samples (1.5 to 6 ng/mL). Using this procedure, we
established differences in GH parameters between boars
and barrows and showed the repeatability of this RIA
method [22,23,25].
Each GH profile of the window samples was
evaluated for pulses. The mean for all 74 samples per
window for each animal was determined. A pulse was
assumed when a GH concentration exceeded this mean
value by at least 50% and was followed by a decrease in
the GH level. The frequency was defined as the number
of pulses per 24 h. The maximal level was the mean of
the maximal concentrations of these pulses. The base
levels were calculated by the mean of the 14 lowest
values.
2.3.3. Insulin-like growth factor-1
Concentrations of IGF-1 were determined by a
double-antibody RIA in plasma samples taken every
second day after HCl/ethanol extraction using a specific
antiserum raised in rabbits [9]. The highly purified IGF-1
standard was obtained from Gro Pep (CU020; Adelaide,
Australia) and used for radio-iodination and for
calibration. Extraction yield was between 61% and
70% for samples spiked from 75 ng/mL to 400 ng/mL
(n = 9). The coefficients of variation within and between
assays were 6.9% and 5.7% for concentrations of 186 ng/
mL (n = 10) and 183 ng/mL (n = 9), respectively.
Fig. 1. Course of GH concentrations (mean + SEM) for the two groups
of barrows (top: early castrated pigs; bottom: late castrated pigs). The
horizontal lines represent the mean values of the two groups.
2.3.4. Urea
Urea in plasma was measured to characterize protein
degradation and therefore nitrogen excretion. Determination was performed on microtiter plates as described
in detail earlier [21]. Intra-assay and interassay
coefficients of variation were 8.9% (n = 10) and 9.7%
(n = 14) at concentrations of 322 mg/mL and 347 mg/
mL, respectively.
Determination was performed as described before
[23]. A specific antiserum (AFP422801) was kindly
provided by Dr. Parlow (NIDDK, Torrance, CA, USA),
and a highly purified pGH (porcine growth hormone)
standard (AFP10864P) was used for radio-iodination
Table 1
Summarized data for GH in window sampling at 19 and 24 wk of age.
Group
Age (wk)
Mean level (ng/mL)
Basal level (ng/mL)
Maximum (ng/mL)
Pulses (n/24 h)
Early castrated
19
Late castrated
24
19
3.5 0.2
P = 0.08
2.8 0.1
3.3 0.2
P < 0.05
2.5 0.2
2.7 0.2
P < 0.05
2.3 0.1
2.6 0.2
P = 0.1
2.1 0.1
6.6 0.4
P < 0.05
4.9 0.2
6.0 0.2
P = 0.08
4.3 0.3
2.5 0.5
P = 0.08
1.3 0.2
2.3 0.7
P = 0.5
1.5 0.4
24
Mean SEM are given. Significance refers to differences between the two windows within one group. Comparison between groups at the same age
revealed no significant differences.
2.3.5. Body fat content and protein synthesis
Total body fat was determined by administration of a
known amount of deuterium oxide via the cephalic vein
catheters. After equilibration, blood samples were
drawn, and deuterium oxide content was measured
by infrared spectrophotometry as described by Claus
et al. [21], and the fat content was calculated.
Protein synthesis was determined by infusion of
[13C]leucine via the cephalic vein catheters at the day of
slaughter, and blood samples were taken over a period
of 6 h. The [13C]leucine to [12C]leucine ratio was
measured via GC-MS (gas chromatography-mass spectrometry) as described elsewhere [22].
2.4. Statistics
Data in the table and figures are presented as
mean SEM of eight animals per group. Data were
analyzed using the Statistical Package for the Social
Sciences (SPSS version 13.0; SPSS Inc., Chicago, IL,
USA). Trend analysis was performed as described in
Refs. [26] and [27]. The mean of the daily samples of each
animal was calculated and used for statistical analysis.
639
Differences between or within groups were tested using
unpaired or paired Student’s t-test, respectively.
3. Results
Testosterone was measured in all animals and
remained below the detection limit of 0.05 ng/mL
between 17 and 29 wk (data not shown). There was no
significant difference between Groups 1 and 2.
Growth hormone concentrations of daily samples are
given in Figure 1. There was no significant increase or
decrease in values (trend analysis). The mean values of
3.04 ng/mL and 3.01 ng/mL for early castrated pigs
(Group 1) and late castrated pigs (Group 2), respectively, also did not differ significantly. Growth hormone
values resulting from evaluation of sampling windows
are summarized in Table 1, and Figure 2 shows
examples for GH profiles for an early- and a latecastrated pig both at 19 and 24 wk of age. Mean values
of windows in Group 2 decreased significantly from 3.3
ng/mL to 2.5 ng/mL from the first window at 19 wk
of age to the second window at 24 wk of age. The
mean values in Group 1 tended to be lower at 24 wk of
Fig. 2. Course of GH concentrations for window sampling for two individual barrows at (left) 19 wk of age and (right) 24 wk of age (top: early
castrated pig; bottom: late castrated pig).
640
Fig. 3. Course of IGF-1 concentrations (mean SEM) for two groups
of barrows (top: early castrated pigs; bottom: late castrated pigs). The
horizontal lines represent the mean values of the two groups.
Fig. 4. Course of urea concentrations (mean SEM) for the two
groups (top: early castrated pigs; bottom: late castrated pigs). The
horizontal lines represent the mean value for the two groups. Low
values at the last day are explained by the experimental design.
age (3.5 ng/mL at 19 wk of age compared with 2.8 ng/mL
at 24 wk of age; P = 0.08). The basal and the maximum
levels were significantly lower in Group 1 compared with
the first and the second window, whereas Group 2 showed
no significant differences although the values tended to
decrease. Nevertheless, there was no significant difference between Group 1 and Group 2 at 19 or 24 wk of age
in any evaluated GH parameter, including pulsatility.
Courses of IGF-1 are presented in Figure 3. Trend
analysis revealed no increase or decrease during the
experimental period. The mean levels were 120 ng/mL
and 123 ng/mL for Groups 1 and 2, respectively, and did
not differ significantly between groups.
Figure 4 gives the urea concentrations for the two
groups. Mean levels were 287 mg/mL for Group 1 and
318 mg/mL for Group 2 and did not differ significantly
between both groups. Trend analysis was also
performed for each animal, but no trend was detected.
Mean total body fat contents at 25 wk of age were
20.1 5.8% and 25.4 7.7% for pigs of Groups 1 and
2, respectively, and showed no significant differences.
Protein synthesis at the day of slaughter revealed
values of 5.44 0.32 g kg-1 d-1 and 6.22 0.44 g kg-1 d-1
for Groups 1 and 2, respectively, which was statistically
significant.
4. Discussion
The GH–IGF-1 system is the main regulator of
anabolic metabolism and growth. It also involves
insulin, which stimulates the expression of GH
receptors in the liver and thus couples IGF-1 secretion
to GH [10,28]. This system is also stimulated by
gonadal steroids. Whereas there is an evident sexual
dimorphism in secretion of GH in rats [29], the situation
in pigs comparing males and females is less obvious [30].
Nevertheless, in sows it was shown that the estrous cycle–
dependent course of estradiol in blood is followed by
concordant changes of GH and IGF-1 [9]. No differences
in IGF-1 were found when comparing intact and
ovariectomized gilts [31]. In boars, testicular steroid
secretion is not only characterized by androgens but also
by estrogens, which may even exceed concentrations in
the follicular phase of the estrous cycle in sows [32].
Thus, the GH–IGF–1 system is set to a higher level
compared with that in females and thus explains sexual
dimorphism. An additional role of androgens is their
anticatabolic effect, which is specifically exerted via
receptors in muscle tissue [33,34]. In other species
(sheep), a testosterone implant failed to alter GH values
in early castrated rams but increased IGF-1 [35].
Because of the practice of castrating male piglets
during the first week of life to avoid later occurrence of
the urine-like ‘‘boar taint’’ in carcasses, the anabolic
effects of gonadal steroids are abolished and the barrows
are inferior in their fattening performance and carcass
composition compared with entire boars [36,37]. Therefore, the mechanisms that differentiate a male pattern of
GH, as it was shown for rats, might be of high practical
relevance when combined with delayed castration (e.g.,
by immunologic techniques) [21,38]. Our data clearly
demonstrated that such a mechanism does not exist in the
pig. This finding is substantiated by Trudeau et al. [39]
who revealed that late castration (6 wk of age) did not
alter GH values significantly.
Interestingly, early castrated pigs showed a lower
protein synthesis compared with that of late castrated
pigs and in parallel a tendency to lower urea excretion
and therefore protein degradation in early castrated
pigs. In consequence, the protein gain for both groups
could be the same. The reason for these findings is
unknown. Additionally, it is likely that intensive care of
the pigs (and thus the avoidance of any stress) during the
leucine infusion period was the reason for decreased
urea concentrations. But again, such influences need
confirmation. The transient postnatal rise of testicular
activity is related to ongoing testicular development
after sexual differentiation early in embryonic life.
Development of pig testes from birth to adulthood is
characterized by two additional waves of development.
One of them is the early postnatal rise of gonadotropins
and testicular steroids around Week 4. These hormones
are also essential to establish Sertoli cells and thus
determine the amount of mature sperm output [40–43].
641
The second postnatal rise occurs around Week 16 and is
usually referred to as the ‘‘final pubertal spurt.’’
In addition, the early postnatal rise of steroidogenesis
in the male pig was suggested to be important for
imprinting later sexual behavior because this rise is
accompanied by mounting attempts of piglets kept in
groups and is supposed to be essential for later normal
sexual behavior in mature boars [44]. Such a mechanism,
however, has to be regarded as a process of learning
instead of differentiation. It was also demonstrated that
differentiation of hypothalamic areas does not play a role,
because male piglets that had been surgically castrated a
few days after birth later exhibited male mounting
behavior when treated with androgens and exhibited
female standing reflex after estradiol application [45,46].
It is concluded that the postnatal rise of steroidogenesis in the male pig does not play a role for a male GH
secretion pattern.
Acknowledgments
We would like to thank Stefanie Mayer, Carmen
Ostertag, and Sybille Knöllinger for their excellent
laboratory work, Bill Dunne, Mohammed Mecellem,
and Claudia Fischinger for care of the animals, and U.
Weiler, who participated in animal surgery. Birgit
Deininger prepared the manuscript. The study was
financially supported by Pfizer Animal Health. We also
thank Michael C. Pearce for valuable discussion and
proofreading of the manuscript.
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General Discussion
3
General Discussion
Immunized boars have a better fattening performance that results in a higher daily gain and
thus a faster growth (Dunshea et al., 2001). This effect or parts of it have been confirmed in
several studies (Jaros et al., 2005; Zamaratskaia et al., 2008a; Andrews et al., 2009; Fuchs
et al., 2009; Hemonic et al., 2009; Pauly et al., 2009; Schmoll et al., 2009; Spring et al.,
2009). The superiority of immunized boars over barrows may be explained by the late onset
of immunization (Turkstra et al., 2002), why immunized boars have the anabolic potential
typical for intact boars for a longer time in the fattening period (Metz and Claus, 2003).
Therefore, the exact time course of titre development and the reaction of hormones and
metabolic parameters are of great importance to optimize the fattening performance of
immunized boars. This determination of reactions before and after the second vaccination
was possible in our studies. Pigs had indwelling vein catheters which allowed daily sampling
of blood samples.
Active immunization against GnRH has a clear effect on the reproductive axis and therefore
on metabolism. Data from our study (see 2.1) once more confirm the effectiveness of
immunization with Improvac®. According to manufacturer´s recommendations, pigs were
immunized twice, 4 weeks apart, and the second vaccination was given 6 weeks prior to
slaughter. In our study, the first vaccination revealed neither an effect on titre development
nor on reproductive hormones. Titres rose within 5 days after the second vaccination. All
animals had high enough titres to release the castration effect. This was confirmed by LH
and testosterone concentrations which decreased within one week. Androstenone
concentrations in plasma decreased the same way and reached basal values of 0.35 ng/mL
within 4-8 days.
The clearance of androstenone from adipose tissue in slaughter weight boars requires
approximately 3-4 weeks (Claus, 1976; Claus, 1979). The rate of clearance depends on age,
e.g. in boars with 240 kg body weight it required 6 weeks to decrease androstenone values
below the threshold of 0.5 ng/g fat. Currently, the recommendation for the timing of the
second vaccination is 4-6 weeks before slaughter. But to achieve an optimal fattening
performance, the immunization schedule should be adapted to the aimed slaughter weight.
Figure 11 shows the weight development of the immunized boars in our studies. The dashed
lines mark 3 different slaughter weights: 80 kg, 100 kg, and 120 kg. For a slaughter weight of
80 kg which was reached with an age of 21 weeks, the second vaccination may be 2 weeks
earlier. For 120 kg slaughter weight, the second vaccination may be in an age of 24-25
weeks, i.e. 3-4 weeks prior to slaughter. In our studies, only a limited number of pigs and
only German Landrace boars were tested. Thus, further studies with a higher animal number
and different breeds are necessary to confirm the timing of the second vaccination before
68
General Discussion
slaughter. A delay in time of slaughter because of different growth rates between the
individual pigs does not compromise the immunization effect. The suppression of testicular
biosynthesis lasted at least 10 weeks after the second immunization (Claus et al., 2008;
Zamaratskaia et al., 2008b).
weight (kg)
120
100
80
60
17
18
19
20
21
22
23
24
25
26
27
28
29 age (weeks)
2. IM
2. IM
2. IM
Figure 11:
Weight development of immunized boars between 18 and 28 weeks of age
(dashed lines mark possible slaughter weights and the corresponding age;
arrows mark second vaccination relative to slaughter, 2. IM)
Another cause for the better fattening performance of immunized boars may be the high GH
concentrations similar to concentrations in intact boars (Metz and Claus, 2003). In the
present study, the maintenance of high levels of GH in the immunized boars could be
additionally confirmed. One mechanism for the elevated GH levels in immunized boars may
be an interaction between the releasing hormones GnRH and GH-RH. Infusion of GnRH led
to an increase of LH and a concomitant decrease of GH, whereas infusion of GH-RH led to
high GH and low LH concentrations (Claus and Weiler, 1994; Weiler, 1995). We investigated
this hypothesis by immunizing barrows (see 2.2). It is known that barrows have high
concentrations of GnRH because of a lack of negative feedback from the testes. If there is a
relation between the two releasing hormones in the hypothalamus or the pituitary, GH-RH
should rise after the immunization because GnRH is blocked. In consequence, GH levels
should also increase thereafter.
The effect of immunization was evident in immunized barrows. Antibody titres rose, and LH
concentrations dropped to levels near the detection limit. But GH concentrations were not
influenced by immunization in barrows. Both groups showed no differences in GH or in IGF-I
69
General Discussion
concentrations. As expected, metabolic parameters did not differ, too. These results do not
exclude a direct interaction between GH-RH and GnRH in the hypothalamus of barrows
which however could not be abolished by immunization, probably because GnRH antibodies
are big molecules which did not reach the hypothalamus so that their effect on GnRH is
restricted to their interaction in blood of the portal veins.
Another explanation for the high GH levels in immunized boars may be an imprinting
mechanism for a male specific GH secretion pattern. In male rats, the GH secretion is
imprinted by a rise of steroids early after birth (Jansson et al., 1985). In pigs, such a rise of
steroids occurs at about 4 weeks of age (Schwarzenberger et al., 1993). This rise cannot
occur in surgically castrated pigs, because castration is routinely performed in the first week
of life. Thus, we compared male pigs which were castrated either at one week of age (before
the rise of testosterone) or at six weeks of age (after the rise of testosterone). But the early
rise of steroids had no effect on GH concentrations (see 2.4). Nevertheless, a male pattern of
GH secretion may exist in the pig. It may be dependent on a transient rise of testicular
steroid synthesis which includes formation of high amounts of oestrogens which were shown
to stimulate GH formation in mature pigs (Claus et al., 1992). In addition, it was found that a
transient rise of testicular activity already occurs in the foetal period (Ford et al., 1980).
The effect of GH on protein synthesis is mediated via IGF-I which is synthesized and
released from the liver (Claus and Weiler, 1994). Thus, high GH concentrations are only
effective for protein synthesis if they are coupled with IGF-I. This GH-IGF-I system is further
influenced by oestrogens and energy availability. Oestrogens (Claus et al., 1992) and
carbohydrates with a high glycaemic index such as starch (Brameld et al., 1999) lead to a
rise of IGF-I concentrations by influencing the GH receptor expression in the liver
(Gabrielsson et al., 1995).
Immunized boars had high GH concentrations, but IGF-I concentrations were lower than in
intact boars (Metz and Claus, 2003). Immunization against GnRH blocks the synthesis of
testicular steroids, and the positive effect of oestrogens on IGF-I is no longer evident.
Consequently, the only influence on IGF-I after the second vacciantion can be via energy
supply through feed allocation. In one study (see 2.3), we investigated the effect of energy
supply through the diet on IGF-I and on metabolic parameters. Boars were immunized twice
according to recommendations. The expected maintenance of high GH concentrations and
the loss of anabolic steroids after the second vaccination were confirmed.
Our results show that pigs fed 3 kg of feed per day had 28% higher IGF-I concentrations in
plasma after the second vaccination compared with pigs fed only 2 kg feed per day. Protein
synthesis was significantly higher in the 3 kg group of immunized boars, whereas protein
70
General Discussion
breakdown did not differ between groups. In consequence, daily weight gain was higher in
the 3 kg group. After the second vaccination, the 3 kg group left no feed residues. Thus, an
elevation of feed consumption may be possible. This may result in a further improvement of
protein synthesis without excessive fat accumulation. Determination of body fat content in the
3 kg group of immunized boars revealed that the fat content is not increased after second
vaccination (13.7% vs 15.0%). Barrows had fat contents of 22% at the same age and under
the same conditions (see 2.2).
The cause of the high GH concentrations in immunized boars could not be identified in our
studies. But results allow an optimal timing of the two vaccinations to get a good
performance of immunized boars without the risk of tainted carcasses at slaughter. In
addition, the influence of feed supply on IGF-I may be another mechanism to optimize the
fattening performance of immunized boars.
Thus, active immunization against GnRH with Improvac® is a practical and reliable
alternative to surgical castration that provides benefits in growth performance and carcass
quality.
71
Summary
4
Summary
Compared to surgical castrates, boars have a superior anabolic potential due to gonadal
steroids, i.e. androgens and oestrogens. In consequence, they have an improved fattening
performance and lean fat ratio in the carcass. However, most male piglets are surgically
castrated without anaesthesia within the first week of life to avoid the unpleasant urine-like
boar taint which is not acceptable to many consumers. Boar taint is mainly caused by
androstenone which is synthesized in the testes together with the gonadal steroids. Skatole
is another compound which contributes to the off odour. But this substance has a faecal
smell and can be controlled by feeding strategies.
Castration without anaesthesia is now regarded to cause acute pain and stress to the piglets,
so that surgical castration is no longer tolerated due to animal welfare considerations.
Different alternatives are discussed but it appears that active immunization against the
gonadotropin releasing hormone (GnRH) is the most practicable alternative to surgical
castration. It is based on application of a commercial antigen (Improvac®) two times at an
interval of 4 weeks. The second vaccination then leads to a high antibody formation and to a
blockade of GnRH and thus luteinizing hormone (LH). In turn, the testicular biosynthesis of
anabolic hormones as well as androstenone is inhibited. Several studies found that
immunized boars still had a better performance than surgical castrates. One explanation is
that immunized boars maintain part of their anabolic potential before the second vaccination
and thus the onset of antibody formation. Therefore, an exact time schedule for immunization
is required to optimize the fattening period without risk of tainted carcasses at slaughter.
Another explanation may be that high growth hormone (GH) concentrations are maintained
in immunized boars at a boar specific level. In addition to low GH, barrows also have low
concentrations of the anabolic insulin-like growth factor I (IGF-I).
The present four studies were performed to investigate the hormonal and metabolic reactions
before and after the second vaccination and to clarify why high GH concentrations are
maintained. In the first study five catheterized boars were immunized twice with Improvac®
at 18 and 22 weeks of age. Antibody titres rose rapidly after the second vaccination and LH
and consequently testosterone concentrations decreased within 5 days. Basal values of
androstenone were reached within 8 day. Ten days after the second vaccination, IGF-I and
urea, which represents protein turnover, had dropped down to levels which are typical for
surgical castrates (barrows). Results from this study confirm that two doses of Improvac® are
effective in inhibiting gonadal steroid production and thus boar taint after the second
72
Summary
vaccination. Studies on maintenance of GH or IGF-I were based on three different
hypotheses.
Hypothesis 1: An interaction between GnRH and growth hormone releasing hormone (GHRH) in the hypothalamus is known from literature. The drop of GnRH due to immunization
might abolish an inhibiting effect on GH-RH and maintenance of GH high concentrations in
immunized boars. In contrast, barrows have high GnRH and LH concentrations due to the
absence of negative feedback by gonadal hormones and thus low GH levels.
Twelve barrows were fitted with an indwelling vein catheter. Six of these barrows were
immunized with Improvac® at 18 and 22 weeks of age. GH, LH, FSH, testosterone and IGF-I
were determined as well as specific parameters to characterize protein synthesis and
degradation, and body fat content. The second vaccination led to high antibody titres and low
levels of LH and testosterone while FSH decreased slowly within 5 weeks to 0.5 ng/mL. GH
as well as IGF-I was not influenced and remained at low concentrations typical for barrows.
Thus, the metabolic status was not altered by immunization and the hypothesis of an
interaction between GnRH and GH-RH could not be substantiated for the pig, probably
because an interaction occurred exclusively in blood of the hypophyseal portal veins but not
in the hypothalamus.
Hypothesis 2: IGF-I is not only stimulated by GH but also by increasing amounts of starch in
the feed. In immunized boars, feed intake is increased due to the absence of gonadal
steroids. Two groups of six immunized boars were given 2 or 3 kg of feed per day. Before the
second vaccination, testicular steroids in blood were normal but decreased rapidly thereafter.
GH concentrations were maintained at high levels after the second immunization. Compared
with the 2 kg group, the 3 kg group showed significant higher IGF-I concentrations, increased
weight gain and increased protein synthesis after the second vaccination. Protein breakdown
did not differ between groups. The body fat content in the 3kg group remained the same
before and after the second vaccination (14% vs. 15%). The results indicate that
performance of immunized boars can be further improved by increasing amount of feed
above current recommendations.
Hypothesis 3: Rodent studies showed that a transient rise of testosterone synthesis about
2 d after birth imprints a later male specific increased secretion of GH. In the male pig, a
transient elevation of testosterone occurs about 4 weeks postnatal. Therefore, a group of
eight male piglets was surgically castrated at 1 week of age, another group of eight male
piglets at 6 weeks of age. All pigs were fitted with catheters at 17 weeks of age. Daily blood
73
Summary
samples were analyzed for testosterone, GH, IGF-I and urea. To determine GH pulsatility
blood was drawn for 24 h every 20 min.
Comparison of the two groups revealed no differences in mean GH concentrations and their
pulsatile secretion pattern. As expected the other metabolic parameter did not differ as well.
It is concluded that the postnatal rise of steroid synthesis in boars is not responsible for the
later pattern of GH secretion in pigs and thus is a rodent specific mechanism.
The cause for elevated GH levels in immunized boars could not be identified in the present
studies. Nevertheless, results allow an exact timing of the vaccinations and thus an optimal
use of the anabolic potential of immunized boars without the risk of tainted carcasses at
slaughter. A further improvement of the fattening performance may be realized by feeding
strategies and their influence on IGF-I.
74
Zusammenfassung
5
Zusammenfassung
Auswirkungen der immunologischen Kastration auf die Stoffwechselregulation beim
Eber
Eber haben im Vergleich zu Kastraten ein höheres anaboles Potential, das durch die
Gonadensteroide (Androgene und Östrogene) bedingt ist. Deshalb weisen sie eine
verbesserte Mastleistung und ein günstigeres Fleisch-Fett-Verhältnis im Schlachtkörper auf.
Die meisten männlichen Ferkel werden dennoch in der ersten Lebenswoche chirurgisch
kastriert, um den urinartigen Ebergeruch zu vermeiden, der von vielen Verbrauchern nicht
akzeptiert wird. Ebergeruch wird hauptsächlich durch Androstenon verursacht, das
zusammen mit den Gonadensteroiden im Hoden gebildet wird. Skatol ist ein weiterer
Bestandteil, der zum Fehlgeruch von Eberfleisch beiträgt. Skatol hat einen fäkalartigen
Geruch und kann aber durch Fütterungsstrategien kontrolliert werden.
Die chirurgische Kastration ohne Betäubung wird mittlerweile als schmerzhaft und belastend
für die Ferkel eingeschätzt. Deshalb wird sie aufgrund des Tierschutzes nicht länger toleriert.
Verschiedene Alternativen werden diskutiert, wobei die aktive Immunisierung gegen
Gonadotropin Releasing Hormone (GnRH) die praktikabelste Alternative zu sein scheint. Die
Immunisierung basiert auf der Anwendung eines kommerziellen Antigens (Improvac®), das
zweimal im Abstand von 4 Wochen verabreicht wird. Die 2. Impfung löst eine hohe
Antikörperbildung aus und führt damit zur Blockade von GnRH und LH. Dies wiederum führt
zur Hemmung der testikulären Biosynthese, d.h. die anabolen Hormone wie auch
Androstenon werden inhibiert. Einigen Studien zufolge weisen immunisierte Eber eine immer
noch bessere Mastleistung als chirurgische Kastraten (Börge) auf. Eine Erklärung dafür ist,
dass immunisierte Eber einen Teil ihres anabolen Potentials vor der 2. Impfung, und damit
vor der Antikörperbildung, erhalten. Deshalb ist ein exakter Zeitplan für die Anwendung des
Impfstoffes nötig, um einerseits die Mastperiode optimal zu gestalten, aber andererseits
keine geruchsbelasteten Schlachtkörper zu riskieren. Eine weitere Erklärung könnten die
hohen Wachstumshormonkonzentrationen (GH) sein, die immunisierte Eber auf einem für
den Eber typischen Niveau aufrechterhalten. Zusätzlich zu niedrigen GH-Konzentrationen,
weisen Börge auch niedrigere Konzentrationen des anabol wirksamen IGF-I auf.
Die vorliegenden vier Studien wurden durchgeführt, um Hormon- und Stoffwechselreaktionen
vor und nach der 2. Impfung zu untersuchen. Weiter sollte geklärt werden, warum hohe GHKonzentrationen aufrecht erhalten werden. In der ersten Studie wurden 5 Eber mit einem
Katheter versehen und zweimal im Alter von 18 und 22 Wochen mit Improvac® geimpft. Der
75
Zusammenfassung
Antikörpertiter stieg nach der 2. Impfung rasch an. Die LH- und folglich auch die
Testosteronkonzentrationen nahmen innerhalb von 5 Tagen ab. Androstenon erreichte
innerhalb von 8 Tagen Basiswerte. 10 Tage nach der 2. Impfung waren IGF-I und Harnstoff,
der den Proteinumsatz darstellt, auf ein Niveau abgefallen, das für Börge charakteristisch ist.
Die Ergebnisse dieser Studie bestätigen, dass 2 Dosierungen von Improvac® die Produktion
der Gonadensteroide effektiv hemmen und daher auch der Ebergeruch nach der 2. Impfung
unterdrückt wird. Die Studien zur Aufrechterhaltung von GH basieren auf
drei
unterschiedlichen Hypothesen.
Hypothese 1: Eine Wechselwirkung im Hypothalamus zwischen GnRH und Growth
Hormone-Releasing Hormone (GH-RH) ist aus der Literatur bekannt. Der Abfall von GnRH
aufgrund der Immunisierung sollte den Hemmeffekt auf GH-RH beseitigen und somit die
hohen GH-Konzentrationen bei immunisierten Ebern erhalten. Im Gegensatz dazu haben
Börge aufgrund des fehlenden negativen Feedbacks der Gonadenhormone hohe
Konzentrationen von GnRH und von LH, was damit zu geringen GH Mengen führt.
12 Börgen wurde ein Venenkatheter verlegt. Sechs dieser Börge wurden im Alter von 18 und
22 Wochen mit Improvac® geimpft. GH, LH, FSH, Testosteron und IGF-I wurden bestimmt,
weiter der Körperfettgehalt und spezifische Parameter, um die Proteinsynthese und den
Proteinabbau zu charakterisieren. Die 2. Impfung führte zu hohen Antikörpertitern und
niedrigen Werten von LH und Testosteron, während FSH langsam innerhalb von 5 Wochen
auf 0,5 ng/mL abnahm. GH wie auch IGF-I wurden nicht beeinflusst und behielten niedrige
Konzentrationen bei, die für Börge typisch sind. Demzufolge wurde der Stoffwechselstatus
durch die Immunisierung nicht verändert und die Hypothese einer Wechselwirkung zwischen
GnRH und GH-RH konnte für das Schwein nicht bewiesen werden. Vermutlich weil eine
Wechselwirkung zwischen den Antikörpern und GnRH nur in Blut der Portalgefäße, nicht
aber im Hypothalamus selbst statt findet.
Hypothese 2: IGF-I wird nicht nur durch GH stimuliert, sondern auch durch zunehmende
Mengen an Stärke im Futter beeinflusst. Immunisierte Eber weisen aufgrund der fehlenden
Gonadensteroide eine höhere Futteraufnahme auf. Zwei Gruppen mit je 6 immunisierten
Ebern erhielten 2 bzw. 3 kg Futter pro Tag. Vor der 2. Impfung waren die
Hodenhormonewerte im Blut normal, nahmen danach aber schnell ab. Die GHKonzentrationen verblieben nach der 2. Impfung auf ihrem hohen Niveau. Im Vergleich mit
der 2 kg-Gruppe wies die 3 kg-Gruppe signifikant höhere IGF-I-Konzentrationen, ein
gesteigertes Wachstum und eine erhöhte Proteinsynthese auf. Der Köperfettgehalt der 3 kgGruppe war vor und nach der 2. Impfung gleich (14% vs.15%). Die Ergebnisse deuten an,
76
Zusammenfassung
dass die Mastleistung der immunisierten Eber weiter verbessert werden kann, indem die
Futtermenge weiter, über die gebräuchlichen Empfehlungen hinaus, erhöht wird.
Hypothese 3: Studien mit Nagetieren zeigten, dass ein vorübergehender Anstieg von
Testosteron 2 Tage nach der Geburt die später männlich spezifische GH-Sekretion prägt.
Beim
männlichen
Schwein
kommt
es
ungefähr
4
Wochen
postnatal
zu
einer
vorübergehenden Erhöhung der Testosteronwerte. Deshalb wurden 8 Ferkel chirurgisch im
Alter von einer Woche kastriert, weitere 8 Ferkel im Alter von 6 Wochen. Alle Schweine
wurden mit 17 Wochen mit einem Katheter versehen. In den täglichen Blutproben wurden
Testosteron, GH, IGF-I und Harnstoff analysiert. Um die Pulsatilität von GH bestimmen zu
können, wurde für 24 h alle 20 min Blutproben genommen. Der Vergleich der 2 Gruppen
ergab keine Unterschiede zwischen den mittleren GH-Konzentrationen und ihrem pulsatilen
Sekretionsmuster. Wie erwartet unterschieden sich die stoffwechselrelevanten Parameter
auch nicht. Folglich ist der postnatale Anstieg der Steroidsynthese beim Eber nicht für das
spätere Sekretionsmuster von GH beim Schwein verantwortlich. Damit kommt dieser
Mechanismus nur bei Nagetieren zum Tragen.
Der Grund für die hohen GH-Konzentrationen bei immunisierten Ebern konnte in den
vorliegenden Studien nicht herausgefunden werden. Dennoch erlauben die Ergebnisse eine
exakte zeitliche Einordnung der Impfungen in die Mastperiode, wodurch das anabole
Potential der immunisierten Eber optimal genutzt werden kann, ohne geruchsbelastete
Schlachtkörper zu riskieren. Eine weitere Verbesserung der Mastleistung könnte durch
Fütterungsstrategien und deren Einfluss auf IGF-I erreicht werden.
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93
Curriculum Vitae
7
Curriculum Vitae
Personal information
Name:
Aneka Bauer
Date of birth:
21.01.1982
Place of birth:
Heilbronn-Neckargartach
Education and working experience
09/1988 - 07/1992
primary school, Unterheinriet
09/1992 - 07/2001
secondary school, Justinus-Kerner-Gymnasium
Heilbronn
qualification: A levels
10/2001 - 09/2006
academic studies in Agricultural Biology with
emphasis in farm animal biology,
University of Hohenheim
qualification: diploma thesis
10/2006-03/2008
scientific employee, Institute of Animal Husbandry and
Animal Breeding, Department of Animal Breeding and
Regulation Physiology, University of Hohenheim
since 09/2009
scientific employee, Max Rubner Institute,
Department of Safety and Quality of Meat, Kulmbach
94
Erklärung
Hiermit erkläre ich, dass ich die vorliegende Dissertation selbstständig angefertigt habe. Es
wurden nur die angegebenen Quellen und Hilfsmittel benutzt, wörtlich oder inhaltlich
übernommene Stellen wurden als solche gekennzeichnet.
Stuttgart-Hohenheim, Juni 2010
Danksagung
Mein aufrichtiger Dank gilt Herrn Prof. Dr. Dr. R. Claus für die Möglichkeit, ein interessantes
und aktuelles Thema zu bearbeiten. Die vielseitige Arbeit am Institut, sowohl praktisch mit
den Tieren als auch die verschiedenen Analysen und Methoden im Labor haben mir ein
breites Betätigungsfeld eröffnet. Weiter möchte ich mich für die Unterstützung und
Anregungen bei den Publikationen und der daraus resultierenden Dissertation bedanken.
Mein weiterer Dank gilt Frau PD Dr. U. Weiler für die Einweisung und die Betreuung bei den
radioimmunologischen Nachweisverfahren der Proteohormone und Ihren Tätigkeiten bei der
Verlegung der Venenverweilkatheter.
Bei Herrn Dr. M. Lacorn bedanke ich mich für seine fachlichen Ratschläge, seine
Unterstützung bei allen Versuchen sowohl in der praktischen Durchführung im Stall, als auch
im Labor.
Bedanken möchte ich mich auch bei allen Mitarbeitern des ehemaligen Fachgebiets für
Tierhaltung und Leistungsphysiologie. Bei den Mitarbeitern im Stall, C. Fischinger, W. Dunne
und M. Mecellem, für die Betreuung der Versuchstiere und bei den Mitarbeitern im Labor, H.
Hägele, S. Knöllinger, S. Mayer und C. Ostertag für ihre Hilfe bei den Analysen. Weiter
danke ich allen nicht namentlich genannten Mitarbeitern sowie Doktoranden, Diplomanden
und Masterstudenten, die mich tatkräftig bei meinen Versuchen und den damit verbundenen
„Nachtschichten“ unterstützt haben und ohne die ich diese nicht in dieser Form hätte
durchführen können.
Der Firma Pfizer Aminal Health danke ich für die finanzielle Unterstützung der
Untersuchungen und die Bereitstellung des Impfstoffes.
Letztlich möchte ich noch meinen Eltern und allen Freunden danken, die mich unterstützt
haben.

Institute of Animal Husbandry and Animal Breeding University of

Transcrição

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